Anti-DEspR inhibitors as therapeutics for inhibition of pathological angiogenesis and tumor cell invasiveness and for molecular imaging and targeted delivery

ABSTRACT

Provided herein are novel compositions comprising anti-DEspR antibodies and fragments thereof, including fully human, composite engineered human, humanized, monoclonal, and polyclonal anto-DEspR antibodies and fragments thereof, and methods of their use in a variety of therapeutic applications. The compositions comprising the anti-DEspR antibodies and fragments thereof described herein are useful in diagnostic and imaging methods, such as DEspR-targeted molecular imaging of angiogenesis, and for companion diagnostic and/or in vivo-non invasive imaging and/or assessments.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application under 35 U.S.C. § 120 ofU.S. Ser. No. 13/811,485 filed Mar. 21, 2013, which is a 35 U.S.C. § 371National Phase Entry Application of International Application No.PCT/US2011/045056 filed 22 Jul. 2011, which designates the U.S., andwhich claims benefit under 35 U.S.C. § 119(e) of U.S. ProvisionalApplication Ser. No. 61/367,206 filed on 23 Jul. 2010, the contents ofwhich are incorporated herein by reference in their entireties.

GOVERNMENT SUPPORT

This invention was made with Government Support Contract No. RR025771awarded by the National Institutes of Health. The Government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Aug. 3, 2017, isnamed 701586-073092 SL.txt and is 31,150 bytes in size.

FIELD OF THE INVENTION

This invention relates to monoclonal antibodies against DEspR, and theiruse as therapeutics in the inhibition of pathological angiogenesis andtumor cell invasiveness, as well as diagnostic agents and targetingagents for molecular imaging and targeted delivery of other therapeuticagents.

BACKGROUND

The establishment of a critical role of the angiogenic switch intumorigenesis has made the rationale behind the development ofanti-angiogenesis therapy clear (Hanahan & Weinberg 2007).Unfortunately, the ability to attain long-term efficacy ofanti-angiogenesis therapy for all cancer-types, in order to reducecancer to a dormant, chronic manageable disease without increasingmorbidity from side effects, has not yet been achieved (Loges et al.2010, Ferrara 2009, Abdollahi & Folkman 2009, Bergers & Hanahan 2008).

Cumulative observations indicate that all three FDA-approved VEGFpathway inhibitors (anti-VEGFbevacizumab or Avastin, AntiVEGFR2sunitinib, and sorafanib) result in only transitory improvements in theform of tumor stasis or shrinkage, and only for certain cancers despitemost, if not all cancer types exhibiting pathological angiogenesis(Carmeliet 2005; Bergers and Hanahan 2008). Moreover, while anti-VEGFpathway therapies have reduced primary tumor growth and metastasis inpreclinical studies (Crawford & Ferrara 2008), recent mouse tumor modelstudies have reported that sunitinib and an anti-VEGFR2 antibody, DC101,increased metastasis of tumor cells despite inhibition of primary tumorgrowth and increased overall survival in some cases (Ebos et al. 2009,Paez-Ribes et al. 2009). Addressing this “antiangiogenesis therapyconundrum,” cumulative observations have suggested several mechanisms ofevasive and intrinsic resistances (Loges et al. 2010, Ferrara 2009,Abdollahi & Folkman 2009, Bergers & Hanahan 2008) such as: a) activationand/or upregulation of alternative pro angiogenic pathways, b)recruitment of bone marrow-derived pro-angiogenic cells, c) increasedpericyte coverage for the tumor vasculature, attenuating the need forVEGF signaling; d) activation and enhancement of invasion and metastasisto provide access to normal tissue vasculature without obligateneovascularization; [for intrinsic resistance]: e) pre-existingmultiplicity of redundant pro-angiogenic signals; f) pre-existinginflammatory cell-mediated vascular protection; g) tumorhypovascularity; and h) invasive and metastatic co-option of normalvessels without requisite angiogenesis (Bergers and Hanahan 2008).

SUMMARY OF THE INVENTION

Described herein are novel compositions comprising anti-DEspR antibodiesand fragments thereof, including fully human, composite engineeredhuman, humanized, monoclonal, and polyclonal anto-DEspR antibodies andfragments thereof, and methods of their use in a variety ofapplications, including, but not limited to: 1) anti-angiogenesistherapies and anti-tumor cell invasiveness relevant for treatment ofcancer, 2) anti-angiogenesis approaches relevant to treatment of thosevascular diseases where pathological angiogenesis plays a role inpathogenesis or progression such as in carotid artery disease, vasavasorum neovascularization (thus impacting stroke), and vulnerableplaque neovascularization (thus impacting, for example, heart disease),and 3) pro-autophagy approaches pertinent to neurodegenerative diseaseswherein increased autophagy can prevent the accumulation of toxicproducts or misfolded proteins or abnormal proteins as in Alzheimer'sdisease, Huntington's disease etc.

In addition, the compositions comprising the anti-DEspR antibodies andfragments thereof described herein are useful in diagnostic and imagingmethods, such as DEspR-targeted molecular imaging of angiogenesis, whichcan be used, for example, in monitoring response to therapy, in vivodetection of tumor “angiogenic switch” or vascular mimicry. Thecompositions comprising the anti-DEspR antibodies and fragments thereofare useful for novel companion diagnostic and/or in vivo-non invasiveimaging and/or assessments. Additionally, the value-added benefit oftargeted delivery of therapeutic agents using the compositionscomprising the anti-DEspR antibodies and fragments thereof is especiallyimportant in cancer wherein maximal efficacy is needed with minimalsystemic toxicity. Notably, such diagnostics provide novel approachesfor anti-angiogenic therapies for use in personalized medicine.Accordingly, the compositions comprising the anti-DEspR antibodies andfragments thereof described herein comprise targeting tools and/ormodules for target-specific delivery of therapeutics, in forms such astoxins, drugs, small molecules, peptides, fusion proteins, chimericproteins, nanoparticles, DNA, siRNA, etc., as well as for combinatorialtarget-specific diagnostics and therapeutics, termed herein as“theranostics.”

Accordingly, provided herein, in some aspects are isolated anti-DEspRantibodies or antibody fragments thereof that specifically bind to DEspR(dual endothelin/VEGF signal peptide receptor) and reduce or inhibitDEspR biological activity.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment thereofspecifically binds to DEspR comprising the amino acid sequence of SEQ IDNO:1. In some embodiments of these aspects, the antibody or antibodyfragment thereof specifically binds to an epitope of DEspR comprisingresidues 1-9 of SEQ ID NO:1. In some embodiments of these aspects, theantibody or antibody fragment thereof specifically binds to an epitopeof DEspR consisting essentially of residues 1-9 of SEQ ID NO: 1. In someembodiments of these aspects, the antibody or antibody fragment thereofspecifically binds to an epitope of DEspR consisting of residues 1-9 ofSEQ ID NO: 1.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment thereofspecifically binds to DEspR at a VEGF signal peptide (VEGFsp) bindingsite. In some such embodiments, the VEGF signal peptide comprises theamino acid sequence of SEQ ID NO:2. In some such embodiments, the VEGFsignal peptide consists essentially of the amino acid sequence of SEQ IDNO:2. In some such embodiments, the VEGF signal peptide consists of theamino acid sequence of SEQ ID NO:2.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody is a monoclonal antibody or antibodyfragment thereof.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment comprises avariable heavy (VH) chain amino acid sequence comprising a sequence ofSEQ ID NO: 4.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment comprises avariable light (VL) chain amino acid sequence comprising a sequence ofSEQ ID NO: 9.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment comprises avariable heavy (VH) chain amino acid sequence comprising a sequence ofSEQ ID NO: 4 and a variable light (VL) chain amino acid sequencecomprising a sequence of SEQ ID NO: 9.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody is a humanized antibody or antibodyfragment thereof.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment comprises one ormore heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. Insome such embodiments, one or more heavy chain CDR regions consistessentially of a sequence selected from the group consisting of SEQ IDNO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some such embodiments, one ormore heavy chain CDR regions consist of a sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment comprises one ormore light chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. Insome such embodiments, one or more light chain CDR regions consistessentially of a sequence selected from the group consisting of SEQ IDNO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some such embodiments, oneor more light chain CDR regions consist of a sequence selected from thegroup consisting of SEQ ID NO: 10-SEQ ID NO: 11, and SEQ ID NO: 12.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment comprises one ormore heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, andone or more light chain CDR regions comprising a sequence selected fromthe group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.In some such embodiments, the one or more heavy chain CDR regionsconsist essentially of a sequence selected from the group consisting ofSEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some such embodiments,the one or more heavy chain CDR regions consist of a sequence selectedfrom the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:7. In some such embodiments, the one or more light chain CDR regionsconsist essentially of a sequence selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some suchembodiments, the one or more light chain CDR regions consist of asequence selected from the group consisting of SEQ ID NO: 10-SEQ ID NO:11, and SEQ ID NO: 12.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody is a composite antibody or antibodyfragment thereof. In some such embodiments, the the anti-DEspR compositeantibody or antibody fragment comprises one or more heavy chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some such embodiments, thethe anti-DEspR composite antibody or antibody fragment comprises one ormore heavy chain CDR regions consisting essentially of a sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, andSEQ ID NO: 7. In some such embodiments, the anti-DEspR compositeantibody or antibody fragment comprises one or more heavy chain CDRregions consisting of a sequence selected from the group consisting ofSEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some such embodiments,the the anti-DEspR composite antibody or antibody fragment comprises oneor more light chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. Insome such embodiments, the the anti-DEspR composite antibody or antibodyfragment comprises one or more light chain CDR regions consistingessentially of a sequence selected from the group consisting of SEQ IDNO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some such embodiments, thethe anti-DEspR composite antibody or antibody fragment comprises one ormore light chain CDR regions consists of a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody is a composite antibody or antibodyfragment thereof comprising a variable heavy (VH) chain amino acidsequence selected from the group consisting of SEQ ID NO: 13-SEQ ID NO:17. In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody is a composite antibody or antibodyfragment thereof consisting essentially of a variable heavy (VH) chainamino acid sequence selected from the group consisting of SEQ ID NO:13-SEQ ID NO: 17. In some embodiments of these aspects and all suchaspects described herein, the anti-DEspR antibody is a compositeantibody or antibody fragment thereof consisting of a variable heavy(VH) chain amino acid sequence selected from the group consisting of SEQID NO: 13-SEQ ID NO: 17.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody is a composite antibody or antibodyfragment thereof comprising a variable light (VL) chain amino acidsequence selected from the group consisting of SEQ ID NO: 18 and SEQ IDNO: 19. In some embodiments of these aspects and all such aspectsdescribed herein, the anti-DEspR antibody is a composite antibody orantibody fragment thereof consisting essentially of a variable light(VL) chain amino acid sequence selected from the group consisting of SEQID NO: 18 and SEQ ID NO: 19. In some embodiments of these aspects andall such aspects described herein, the anti-DEspR antibody is acomposite antibody or antibody fragment thereof consisting of a variablelight (VL) chain amino acid sequence selected from the group consistingof SEQ ID NO: 18 and SEQ ID NO: 19.

In other embodiments of these aspects, the anti-DEspR antibody orantibody fragment thereof is an antibody expressed or produced byhybridomas 7C5C55 or G12E8.

In some embodiments of these aspects, the anti-DEspR antibody orantibody fragment thereof displays a similar binding pattern to thebinding pattern displayed by an antibody expressed or produced byhybridomas 7C5B2, 7C5C55, or G12E8. In some embodiments of theseaspects, the anti-DEspR antibody or antibody fragment thereof displays asimilar avidity to the avidity displayed by an antibody expressed orproduced by hybridomas 7C5B2, 7C5C55, or G12E8. In some embodiments ofthese aspects, the anti-DEspR antibody or antibody fragment thereofbinds to the same epitope(s) as those epitope(s) bound by an antibodyexpressed or produced by hybridomas 7C5B2, 7C5C55, or G12E8.

In some embodiments of these aspects, the anti-DEspR antibody orantibody fragment thereof comprises an amino acid sequence of one ormore CDRs of an antibody expressed or produced by hybridomas 7C5C55 orG12E8. In some embodiments of these aspects, the anti-DEspR antibody orantibody fragment thereof has one or more biological characteristics ofa monoclonal antibody expressed or produced by hybridoma 7C5B2, 7C5C55,or G12E8. In some embodiments of these aspects, the anti-DEspR antibodyor antibody fragment thereof specifically binds to an epitope of DEspRthat is bound by an antibody expressed or produced by hybridoma 7C5B2,7C5C55, or G12E8.

In some embodiments of these aspects and all such aspects describedherein, the antibody fragment is a Fab fragment, a Fab' fragment, a Fdfragment, a Fd' fragment, a Fv fragment, a dAb fragment, a F(ab')2fragment, a single chain fragment, a diabody, or a linear antibody.

In some embodiments of these aspects and all such aspects describedherein, the anti-DEspR antibody or antibody fragment thereof furthercomprises an agent conjugated to the anti-DEspR antibody or antibodyfragment thereof to form an immunoconjugate specific for DEspR. In somesuch embodiments, the agent conjugated to the antibody or antibodyfragment thereof is a chemotherapeutic agent, a toxin, a radioactiveisotope, a small molecule, an siRNA, a nanoparticle, or a microbubble.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR, and apharmaceutically acceptable carrier.

In some aspects, provided herein are methods of inhibiting angiogenesisin a subject having a disease or disorder dependent or modulated byangiogenesis, comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR. In someembodiments of these aspects and all such aspects described herein, thedisease or disorder dependent or modulated by angiogenesis is a canceror a tumor. In some embodiments of these aspects and all such aspectsdescribed herein, the disease or disorder dependent or modulated byangiogenesis is selected from the group consisting of age-relatedmacular degeneration, carotid artery disease, diabetic retinopathy,rheumatoid arthritis, neurodegenerative disorder, Alzheimer's disease,obesity, endometriosis, psoriasis, atherosclerosis, ocularneovascularization, neovascular glaucoma, osteoporsosis, and restenosis.

In some aspects, provided herein are methods of inhibiting tumor cellinvasiveness in a subject having a cancer or a tumor, comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising any of the anti-DEspRantibodies or antibody fragments thereof described herein thatspecifically binds to DEspR. In some embodiments of these aspects andall such aspects described herein, the method further comprises theadministration of one or more chemotherapeutic agents, angiogenesisinhibitors, cytotoxic agents, or anti-proliferative agents.

In some aspects, provided herein are methods of inhibiting tumor growthand reducing tumor size or tumor metastasis in a subject having a tumoror metastasis by inhibiting DEspR expression and/or function in a cell,comprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising any of theanti-DEspR antibodies or antibody fragments thereof described hereinthat specifically binds to DEspR. In some embodiments of these aspectsand all such aspects described herein, the DEspR expression and/orfunction is inhibited in a tumor cell, a tumor initiating cell, a cancerstem-like cell, a cancer stem cell, a metastatic tumor cell, anendothelial progenitor cell, an inflammatory cell, a tumor stromal cell,a tumor vasculature cell, or any combination thereof. In some suchembodiments, the tumor vasculature cell is an endothelial cell, apericyte, a smooth muscle cell, an adventitial cell, or any combinationthereof.

In some aspects, provided herein are methods of inhibiting tumorresistance and tumor recurrence in a subject by inhibiting DEspRexpression and/or function in a cell, the methods comprisingadministering to a subject in need thereof a therapeutically effectiveamount of a pharmaceutical composition comprising any of the anti-DEspRantibodies or antibody fragments thereof described herein thatspecifically binds to DEspR. In some embodiments of these aspects andall such aspects described herein, the DEspR expression and/or functionis inhibited in a tumor cell, a tumor initiating cell, a cancerstem-like cell, a cancer stem cell, a metastatic tumor cell, or anycombination thereof.

In some aspects, provided herein are methods of inhibiting cancerprogression through promotion of autophagy of a cancer cell byinhibiting DEspR expression and/or function in a tumor cell, the methodscomprising administering to a subject in need thereof a therapeuticallyeffective amount of a pharmaceutical composition comprising any of theanti-DEspR antibodies or antibody fragments thereof described hereinthat specifically binds to DEspR. In some embodiments of these aspectsand all such aspects described herein, the DEspR expression and/orfunction is inhibited in a tumor cell, a tumor initiating cell, a cancerstem-like cell, a cancer stem cell, a metastatic tumor cell, or anycombination thereof.

In some aspects, provided herein are methods of promoting autophagy or areduction in accumulation of intracellular noxious substances orpathogens by inhibiting DEspR expression and/or function in a cell, themethods comprising administering to a subject in need thereof atherapeutically effective amount of a pharmaceutical compositioncomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR. In someembodiments of these aspects and all such aspects described herein, thesubject has Alzheimer's disease or Huntington's disease.

In some aspects, provided herein are methods of molecular imaging viatargeting DEspR, the methods comprising administering an effectiveamount of a pharmaceutical composition comprising any of the anti-DEspRantibodies or antibody fragments thereof described herein thatspecifically binds to DEspR conjugated to a targeting moiety, anddetermining the presence or absence of the anti-DEspR antibodies orantibody fragments thereof conjugated to the targeting moiety usingmolecular imaging. In some embodiments of these aspects and all suchaspects described herein, the molecular imaging is contrast-enhancedultrasound imaging, MRI (magnetic resonance imaging), near infraredimaging, or photoacoustics imaging. In some embodiments of these aspectsand all such aspects described herein, the targeting moiety is anantibody, a DEspR-binding peptide ligand, a small molecule, ananoparticle, a polymer, an aptamer, or any combination thereof.

In some aspects, provided herein are methods of stratifying orclassifying a tumor via determination of DEspR expression, the methodscomprising contacting a cell with any of the anti-DEspR antibodies orantibody fragments thereof described herein that specifically binds toDEspR, and determining whether the anti-DEspR antibody or antibodyfragment thereof binds to the cell after said contacting, such thatbinding of the DEspR antibody or antibody fragment thereof to the cellindicates that the cell expresses DEspR. In some embodiments of theseaspects and all such aspects described herein, the cell is a tumor cell,an endothelial cell, a pericyte, a smooth muscle cell, an adventitialcell, a tumor stromal cell, or any combination thereof. In some suchembodiments, the tumor stromal cell is a fibroblast, a myofibroblast, aninflammatory cell, a stellate cell, or any combination thereof. In someembodiments of these aspects and all such aspects described herein, thecell being contacted is in a tissue biopsy, a paraffin-embedded section,or a frozen section.

In some aspects, provided herein are methods for enhancing delivery of atherapeutic agent via DEspR-targeted sonoporation, the methodscomprising delivering an effective amount of a pharmaceuticalcomposition comprising any of the anti-DEspR antibodies or antibodyfragments thereof described herein that specifically binds to DEspR anda therapeutic agent using targeted ultrasound delivery, to a subject inneed thereof, such that delivery of the therapeutic agent is enhanced orincreased relative to delivering the therapeutic agent in the absence ofthe pharmaceutical composition comprising any of the anti-DEspRantibodies or antibody fragments thereof described herein. In someembodiments of these aspects and all such aspects described herein, thetherapeutic agent is a chemotherapeutic agent, a small molecule, apeptide, or an aptamer.

Also provided herein, in some aspects, are method for reducing toxicityof a therapeutic agent via DEspR-targeted sonoporation, the methodscomprising delivering an effective amount of a pharmaceuticalcomposition comprising any of the anti-DEspR antibodies or antibodyfragments thereof described herein that specifically binds to DEspR anda therapeutic agent using targeted ultrasound delivery to a subject inneed thereof, such that toxicity of the therapeutic agent is reducedrelative to delivering the therapeutic agent in the absence of thepharmaceutical composition comprising any of the anti-DEspR antibodiesor antibody fragments thereof described herein. In some embodiments ofthese aspects and all such aspects described herein, the therapeuticagent is a chemotherapeutic agent, a small molecule, a peptide, or anaptamer.

In some aspects, provided herein are methods for combiningDEspR-targeted molecular imaging and DEspR-targeted delivery of atherapeutic agent. These methods comprise administering to a subject aneffective amount of a therapeutic agent and a pharmaceutical compositioncomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein conjugated to a targeting moiety, anddetermining the presence or absence of the anti-DEspR antibodies orantibody fragments thereof described herein conjugated to the targetingmoiety using molecular imaging. In some embodiments of these aspects andall such aspects described herein, the molecular imaging iscontrast-enhanced ultrasound imaging, MRI (magnetic resonance imaging),near infrared imaging, or photoacoustics imaging. In some embodiments ofthese aspects and all such aspects described herein, the therapeuticagent is a chemotherapeutic agent, a small molecule, a peptide, or anaptamer.

In other aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use ininhibiting angiogenesis in a subject having a disease or disorderdependent or modulated by angiogenesis. In some embodiments of theseaspects and all such aspects described herein, the disease or disorderdependent or modulated by angiogenesis is a cancer or a tumor. In someembodiments of these aspects and all such aspects described herein, thedisease or disorder dependent or modulated by angiogenesis is selectedfrom the group consisting of age-related macular degeneration, carotidartery disease, diabetic retinopathy, rheumatoid arthritis,neurodegenerative disorder, Alzheimer's disease, obesity, endometriosis,psoriasis, atherosclerosis, ocular neovascularization, neovascularglaucoma, osteoporsosis, and restenosis.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use ininhibiting tumor cell invasiveness in a subject having a cancer or atumor. In some embodiments of these aspects and all such aspectsdescribed herein, the pharmaceutical compositions further comprise oneor more chemotherapeutic agents, angiogenesis inhibitors, cytotoxicagents, or anti-proliferative agents.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use ininhibiting tumor growth and reducing tumor size or tumor metastasis byinhibiting DEspR expression and/or function in a cell in a subject inneed thereof. In some embodiments of these aspects and all such aspectsdescribed herein, the DEspR expression and/or function is inhibited in atumor cell, a tumor initiating cell, a cancer stem-like cell, a cancerstem cell, a metastatic tumor cell, an endothelial progenitor cell, aninflammatory cell, a tumor stromal cell, a tumor vasculature cell, orany combination thereof. In some such embodiments, the tumor vasculaturecell is an endothelial cell, a pericyte, a smooth muscle cell, anadventitial cell, or any combination thereof.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use ininhibiting tumor resistance and tumor recurrence by inhibiting DEspRexpression and/or function in a cell in a subject in need thereof. Insome embodiments of these aspects and all such aspects described herein,the DEspR expression and/or function is inhibited in a tumor cell, atumor initiating cell, a cancer stem-like cell, a cancer stem cell, ametastatic tumor cell, or any combination thereof.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use ininhibiting cancer progression through promotion of autophagy of a cancercell by inhibiting DEspR expression and/or function in a tumor cell in asubject in need thereof. In some embodiments of these aspects and allsuch aspects described herein, the DEspR expression and/or function isinhibited in a tumor cell, a tumor initiating cell, a cancer stem-likecell, a cancer stem cell, a metastatic tumor cell, or any combinationthereof.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use inpromoting autophagy or a reduction in accumulation of intracellularnoxious substances or pathogens by inhibiting DEspR expression and/orfunction in a subject in need thereof. In some embodiments of theseaspects and all such aspects described herein, the subject hasAlzheimer's disease or Huntington's disease.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use inenhancing delivery of a therapeutic agent via DEspR-targetedsonoporation using targeted ultrasound delivery to a subject in needthereof. In some embodiments of these aspects and all such aspectsdescribed herein, the therapeutic agent is a chemotherapeutic agent, asmall molecule, a peptide, or an aptamer.

In some aspects, provided herein are pharmaceutical compositionscomprising any of the anti-DEspR antibodies or antibody fragmentsthereof described herein that specifically binds to DEspR for use inreducing toxicity of a therapeutic agent via DEspR-targeted sonoporationusing targeted ultrasound delivery to a subject in need thereof. In someembodiments of these aspects and all such aspects described herein, thetherapeutic agent is a chemotherapeutic agent, a small molecule, apeptide, or an aptamer.

Definitions

A “DEspR antagonist” refers to a molecule capable of neutralizing,blocking, inhibiting, abrogating, reducing or interfering with DEspRactivities including its binding to endothelin-1 or VEGFsp. DEspRantagonists include anti-DEspR antibodies and antigen-binding fragmentsthereof, receptor molecules and derivatives that bind specifically toDEspR thereby inhibiting, preventing, or sequestering its binding to itsligands, such as VEGFsp and endothelin-1.

The term “antibody” is used in the broadest sense and includesmonoclonal antibodies (including full length or intact monoclonalantibodies), polyclonal antibodies, multivalent antibodies,multispecific antibodies (e.g., bispecific antibodies), and antibodyfragments (see below) so long as they exhibit the desired biologicalactivity and specificity.

As used herein, the term “target” refers to a biological molecule (e.g.,peptide, polypeptide, protein, lipid, carbohydrate) to which apolypeptide domain which has a binding site can selectively bind. Thetarget can be, for example, an intracellular target (e.g., anintracellular protein target) or a cell surface target (e.g., a membraneprotein, a receptor protein). Preferably, a target is a cell surfacetarget, such as a cell surface protein.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which an antibody or antibodyfragment thereof as described herein can bind. The specificity of anantibody or antibody fragment thereof can be determined based onaffinity and/or avidity. The affinity, represented by the equilibriumconstant for the dissociation (K_(D)) of an antigen with anantigen-binding protein, is a measure of the binding strength between anantigenic determinant and an antigen-binding site on the antigen-bindingprotein, such as an antibody or antibody fragment thereof: the lesserthe value of the K_(D), the stronger the binding strength between anantigenic determinant and the antigen-binding molecule. Alternatively,the affinity can also be expressed as the affinity constant (K_(A)),which is 1/K_(D)). As will be clear to the skilled person, affinity canbe determined in a manner known per se, depending on the specificantigen of interest. Accordingly, an antibody or antibody fragmentthereof as defined herein is said to be “specific for” a first target orantigen compared to a second target or antigen when it binds to thefirst antigen with an affinity (as described above, and suitablyexpressed, for example as a K_(D) value) that is at least 10 times, suchas at least 100 times, and preferably at least 1000 times, and up to10000 times or more better than the affinity with which said amino acidsequence or polypeptide binds to another target or polypeptide.

Avidity is the measure of the strength of binding between anantigen-binding molecule (such as an antibody or antibody fragmentthereof described herein) and the pertinent antigen. Avidity is relatedto both the affinity between an antigenic determinant and its antigenbinding site on the antigen-binding molecule, and the number ofpertinent binding sites present on the antigen-binding molecule.Typically, antigen-binding proteins (such as an antibody or antibodyfragment thereof described herein) will bind to their cognate orspecific antigen with a dissociation constant (K_(D) of 10⁻⁵ to 10⁻¹²moles/liter or less, and preferably 10⁻⁷ to 10⁻¹² moles/liter or lessand more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e., with an associationconstant (K_(A)) of 10⁵ to 10¹² liter/moles or more, and preferably 10⁷to 10¹² liter/moles or more and more preferably 10⁸ to 10¹²liter/moles). Any K_(D) value greater than 10⁻⁴ mol/liter (or any K_(A)value lower than 10⁴ M⁻¹) is generally considered to indicatenon-specific binding. The K_(D) for biological interactions which areconsidered meaningful (e.g., specific) are typically in the range of10⁻¹⁰ M (0.1 nM) to 10⁻⁵ M (10000 nM). The stronger an interaction is,the lower is its K_(D). Preferably, a binding site on an antibody orantibody fragment thereof described herein will bind to the desiredantigen with an affinity less than 500 nM, preferably less than 200 nM,more preferably less than 10 nM, such as less than 500 pM. Specificbinding of an antigen-binding protein to an antigen or antigenicdeterminant can be determined in any suitable manner known per se,including, for example, Scatchard analysis and/or competitive bindingassays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) andsandwich competition assays, and the different variants thereof knownper se in the art; as well as other techniques as mentioned herein.

Accordingly, as used herein, “selectively binds” or “specifically binds”refers to the ability of an antibody or antibody fragment thereofdescribed herein to bind to a target, such as a molecule present on thecell-surface, with a K_(D) 10⁻⁵ M (10000 nM) or less, e.g., 10⁻⁶ M, 10⁻⁷M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰M, 10⁻¹¹ M, 10⁻¹² M, or less. Specific bindingcan be influenced by, for example, the affinity and avidity of thepolypeptide agent and the concentration of polypeptide agent. The personof ordinary skill in the art can determine appropriate conditions underwhich the polypeptide agents described herein selectively bind thetargets using any suitable methods, such as titration of a polypeptideagent in a suitable cell binding assay.

As described herein, an “antigen” is a molecule that is bound by abinding site on a polypeptide agent, such as an antibody or antibodyfragment thereof. Typically, antigens are bound by antibody ligands andare capable of raising an antibody response in vivo. An antigen can be apolypeptide, protein, nucleic acid or other molecule. In the case ofconventional antibodies and fragments thereof, the antibody binding siteas defined by the variable loops (L1, L2, L3 and H1, H2, H3) is capableof binding to the antigen. The term “antigenic determinant” refers to anepitope on the antigen recognized by an antigen-binding molecule, andmore particularly, by the antigen-binding site of said molecule.

As used herein, an “epitope” can be formed both from contiguous aminoacids, or noncontiguous amino acids juxtaposed by tertiary folding of aprotein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents, whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5, about 9, or about 8-10 amino acids in a unique spatialconformation. An “epitope” includes the unit of structure conventionallybound by an immunoglobulin V_(H)/V_(L) pair. Epitopes define the minimumbinding site for an antibody, and thus represent the target ofspecificity of an antibody. In the case of a single domain antibody, anepitope represents the unit of structure bound by a variable domain inisolation. The terms “antigenic determinant” and “epitope” can also beused interchangeably herein.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that can be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen. Themodifier “monoclonal” is not to be construed as requiring production ofthe antibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention can be made bythe hybridoma method first described by Kohler et al., Nature 256:495(1975), or can be made by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). The “monoclonal antibodies” can also be isolated fromphage antibody libraries using the techniques described in Clackson etal., Nature 352:624-628 (1991) or Marks et al., J. Mol. Biol.222:581-597 (1991), for example. A monoclonal antibody can be of anyspecies, including, but not limited to, mouse, rat, goat, rabbit, andhuman monoclonal antibodies.

The term “antibody fragment,” as used herein, refer to a proteinfragment that comprises only a portion of an intact antibody, generallyincluding an antigen binding site of the intact antibody and thusretaining the ability to bind antigen. Examples of antibody fragmentsencompassed by the present definition include: (i) the Fab fragment,having V_(L), C_(L), V_(H) and C_(H)1 domains; (ii) the Fab' fragment,which is a Fab fragment having one or more cysteine residues at theC-terminus of the C_(H)1 domain; (iii) the Fd fragment having V_(H) andC_(H)1 domains; (iv) the Fd' fragment having V_(H) and C_(H)1 domainsand one or more cysteine residues at the C-terminus of the CH1 domain;(v) the Fv fragment having the V_(L) and V_(H) domains of a single armof an antibody; (vi) the dAb fragment (Ward et al., Nature 341, 544-546(1989)) which consists of a V_(H) domain; (vii) isolated CDR regions;(viii) F(ab')₂ fragments, a bivalent fragment including two Fab′fragments linked by a disulphide bridge at the hinge region; (ix) singlechain antibody molecules (e.g., single chain Fv; scFv) (Bird et al.,Science 242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883(1988)); (x) “diabodies” with two antigen binding sites, comprising aheavy chain variable domain (V_(H)) connected to a light chain variabledomain (V_(L)) in the same polypeptide chain (see, e.g., EP 404,097; WO93/11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); (xi) “linear antibodies” comprising a pair of tandem Fdsegments (V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementarylight chain polypeptides, form a pair of antigen binding regions (Zapataet al. Protein Eng. 8(10):1057-1062 (1995); and U.S. Pat. No.5,641,870).

An “Fv” fragment is an antibody fragment which contains a completeantigen recognition and binding site. This region consists of a dimer ofone heavy and one light chain variable domain in tight association,which can be covalent in nature, for example in scFv. It is in thisconfiguration that the three CDRs of each variable domain interact todefine an antigen binding site on the surface of the V_(H)-V_(L) dimerCollectively, the six CDRs or a subset thereof confer antigen bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three CDRs specific for an antigen) hasthe ability to recognize and bind antigen, although usually at a loweraffinity than the entire binding site.

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), which is also available on the world wide web, and is expresslyincorporated herein in its entirety by reference. The “EU index as inKabat” refers to the residue numbering of the human IgG1 EU antibody.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; i.e., CDR1,CDR2, and CDR3), and Framework Regions (FRs). V_(H) refers to thevariable domain of the heavy chain. V_(L) refers to the variable domainof the light chain. According to the methods used in this invention, theamino acid positions assigned to CDRs and FRs can be defined accordingto Kabat (Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md., 1987 and 1991)) Amino acidnumbering of antibodies or antigen binding fragments is also accordingto that of Kabat.

As used herein, the term “Complementarity Determining Regions” (CDRs),i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding. Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region cancomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e., about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e., about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someinstances, a complementarity determining region can include amino acidsfrom both a CDR region defined according to Kabat and a hypervariableloop. For example, the CDRH1 of the heavy chain of antibody 4D5 includesamino acids 26 to 35.

Framework regions” (hereinafter FR) are those variable domain residuesother than the CDR residues. Each variable domain typically has four FRsidentified as FR1, FR2, FR3 and FR4. If the CDRs are defined accordingto Kabat, the light chain FR residues are positioned at about residues1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and theheavy chain FR residues are positioned about at residues 1-30 (HCFR1),36-49 (HCFR2), 66-94 (HCFR3), and 103-113 (HCFR4) in the heavy chainresidues. If the CDRs comprise amino acid residues from hypervariableloops, the light chain FR residues are positioned about at residues 1-25(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the lightchain and the heavy chain FR residues are positioned about at residues1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in theheavy chain residues. In some instances, when the CDR comprises aminoacids from both a CDR as defined by Kabat and those of a hypervariableloop, the FR residues will be adjusted accordingly. For example, whenCDRH1 includes amino acids H26-H35, the heavy chain FR1 residues are atpositions 1-25 and the FR2 residues are at positions 36-49.

The “Fab” fragment contains a variable and constant domain of the lightchain and a variable domain and the first constant domain (C_(H)1) ofthe heavy chain. F(ab') ₂ antibody fragments comprise a pair of Fabfragments which are generally covalently linked near their carboxytermini by hinge cysteines between them. Other chemical couplings ofantibody fragments are also known in the art.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally the Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

The expression “linear antibodies” refers to the antibodies described inZapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, theseantibodies comprise a pair of tandem Fd segments(V_(H)-C_(H)1-V_(H)-C_(H)1) which, together with complementary lightchain polypeptides, form a pair of antigen binding regions. Linearantibodies can be bispecific or monospecific.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567;and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that are engineered or designed to comprise minimal sequencederived from non-human immunoglobulin. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a hypervariable region of the recipient are replaced byresidues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat, rabbit or nonhuman primate having thedesired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human immunoglobulin are replacedby corresponding non-human residues. Furthermore, humanized antibodiescan comprise residues which are not found in the recipient antibody orin the donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FR regions are those of a human immunoglobulin sequence. Thehumanized antibody optionally also will comprise at least a portion ofan immunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). As used herein, a“composite human antibody” is a specific type of engineered or humanizedantibody.

A “human antibody,” “non-engineered human antibody,” or “fully humanantibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art. In one embodiment, the human antibody is selected froma phage library, where that phage library expresses human antibodies(Vaughan et al. Nature Biotechnology 14:309-314 (1996): Sheets et al.Proc. Natl. Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)). Human antibodies can also be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous mouse immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg andHuszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the humanantibody can be prepared via immortalization of human B lymphocytesproducing an antibody directed against a target antigen (such Blymphocytes can be recovered from an individual or can have beenimmunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies andCancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol,147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result an improvement in the affinity ofthe antibody for antigen, compared to a parent antibody which does notpossess those alteration(s). Preferred affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by V_(H) and V_(L) domain shuffling. Random mutagenesis ofCDR and/or framework residues is described by: Barbas et al. Proc Nat.Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al.,J. Immunol 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol.226:889-896 (1992).

A “functional antigen binding site” of an antibody is one which iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same. For multimericantibodies, the number of functional antigen binding sites can beevaluated using ultracentrifugation analysis as described in Example 2of U.S. Patent Application Publication No. 20050186208. According tothis method of analysis, different ratios of target antigen tomultimeric antibody are combined and the average molecular weight of thecomplexes is calculated assuming differing numbers of functional bindingsites. These theoretical values are compared to the actual experimentalvalues obtained in order to evaluate the number of functional bindingsites.

As used herein, a “blocking” antibody or an antibody “antagonist” is onewhich inhibits or reduces biological activity of the antigen it binds.For example, a DEspR -specific antagonist antibody binds DEspR andinhibits the ability of DEspR to, for example, bind VEGFsp and induceangiogenesis, to induce vascular endothelial cell proliferation or toinduce vascular permeability. In certain embodiments, blockingantibodies or antagonist antibodies completely inhibit the biologicalactivity of the antigen.

Unless indicated otherwise, the expression “multivalent antibody” isused throughout this specification to denote an antibody comprisingthree or more antigen binding sites. For example, the multivalentantibody is engineered to have the three or more antigen binding sitesand is generally not a native sequence IgM or IgA antibody.

An antibody having a “biological characteristic” of a designatedantibody is one which possesses one or more of the biologicalcharacteristics of that antibody which distinguish it from otherantibodies that bind to the same antigen.

In order to screen for antibodies which bind to an epitope on an antigenbound by an antibody of interest, a routine cross-blocking assay such asthat described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen(i.e., has a binding affinity (K_(D)) value of no more than about1×10⁻⁷M, preferably no more than about 1X10⁻⁸ M and most preferably nomore than about 1×10⁻⁹ M) but has a binding affinity for a homologue ofthe antigen from a second nonhuman mammalian species which is at leastabout 50 fold, or at least about 500 fold, or at least about 1000 fold,weaker than its binding affinity for the human antigen. Thespecies-dependent antibody can be any of the various types of antibodiesas defined above, but typically is a humanized or human antibody.

As used herein, “antibody mutant” or “antibody variant” refers to anamino acid sequence variant of the species-dependent antibody whereinone or more of the amino acid residues of the species-dependent antibodyhave been modified. Such mutants necessarily have less than 100%sequence identity or similarity with the species-dependent antibody. Inone embodiment, the antibody mutant will have an amino acid sequencehaving at least 75% amino acid sequence identity or similarity with theamino acid sequence of either the heavy or light chain variable domainof the species-dependent antibody, more preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, and mostpreferably at least 95%. Identity or similarity with respect to thissequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e., same residue) orsimilar (i e , amino acid residue from the same group based on commonside-chain properties, see below) with the species-dependent antibodyresidues, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. None ofN-terminal, C-terminal, or internal extensions, deletions, or insertionsinto the antibody sequence outside of the variable domain shall beconstrued as affecting sequence identity or similarity.

To increase the half-life of the antibodies or polypeptide containingthe amino acid sequences described herein, one can attach a salvagereceptor binding epitope to the antibody (especially an antibodyfragment), as described, e.g., in U.S. Pat. No. 5,739,277. For example,a nucleic acid molecule encoding the salvage receptor binding epitopecan be linked in frame to a nucleic acid encoding a polypeptide sequencedescribed herein so that the fusion protein expressed by the engineerednucleic acid molecule comprises the salvage receptor binding epitope anda polypeptide sequence described herein. As used herein, the term“salvage receptor binding epitope” refers to an epitope of the Fc regionof an IgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsiblefor increasing the in vivo serum half-life of the IgG molecule (e.g.,Ghetie et al., Ann. Rev. Immunol 18:739-766 (2000), Table 1). Antibodieswith substitutions in an Fc region thereof and increased serumhalf-lives are also described in WO00/42072, WO 02/060919; Shields etal., J. Biol. Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem.279:6213-6216 (2004)). In another embodiment, the serum half-life canalso be increased, for example, by attaching other polypeptidesequences. For example, antibodies or other polypeptides useful in themethods of the invention can be attached to serum albumin or a portionof serum albumin that binds to the FcRn receptor or a serum albuminbinding peptide so that serum albumin binds to the antibody orpolypeptide, e.g., such polypeptide sequences are disclosed inWO01/45746. In one embodiment, the serum albumin peptide to be attachedcomprises an amino acid sequence of DICLPRWGCLW (SEQ ID NO:3). Inanother embodiment, the half-life of a Fab is increased by thesemethods. See also, Dennis et al. J. Biol. Chem. 277:35035-35043 (2002)for additional serum albumin binding peptide sequences.

A “chimeric DEspR receptor protein” is a DEspR molecule having aminoacid sequences derived from at least two different proteins, at leastone of which is a DEspR protein. In certain embodiments, the chimericDEspR protein is capable of binding to and inhibiting the biologicalactivity of DEspR.

An “isolated” antibody is one that has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould interfere with diagnostic or therapeutic uses for the antibody,and can include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In certain embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined by,for example, the Lowry method, and most preferably more than 99% byweight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cupsequenator, or (3) to homogeneity by SDS-PAGE under reducing ornonreducing conditions using Coomassie blue or, silver stain. Isolatedantibody includes the antibody in situ within recombinant cells since atleast one component of the antibody's natural environment will not bepresent. Ordinarily, however, isolated antibody will be prepared by atleast one purification step.

By “fragment” is meant a portion of a polypeptide, such as an antibodyor antibody fragment thereof, or nucleic acid molecule that contains,preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or more of the entire length of the reference nucleic acid molecule orpolypeptide. A fragment can contain 10, 20, 30, 40, 50, 60, 70, 80, 90,or 100, 200, 300, 400, 500, 600, or more nucleotides or 10, 20, 30, 40,50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 190, 200 amino acids ormore.

An “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to asmall molecular weight substance, a polynucleotide, a polypeptide, anisolated protein, a recombinant protein, an antibody, or conjugates orfusion proteins thereof, that inhibits angiogenesis, vasculogenesis, orundesirable vascular permeability, either directly or indirectly. Itshould be understood that the anti-angiogenesis agent includes thoseagents that bind and block the angiogenic activity of the angiogenicfactor or its receptor. For example, an anti-angiogenesis agent is anantibody or other antagonist to an angiogenic agent as definedthroughout the specification or known in the art, e.g., but are notlimited to, antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDRreceptor or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such asGleevec™ (Imatinib Mesylate). Anti-angiogensis agents also includenative angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See,e.g., Klagsbrun and D′Amore, Annu. Rev. Physiol., 53:217-39 (1991);Streit and Detmar, Oncogene, 22:3172-3179 (2003) (e.g., Table 3 listinganti-angiogenic therapy in malignant melanoma); Ferrara & Alitalo,Nature Medicine 5:1359-1364 (1999); Tonini et al., Oncogene,22:6549-6556 (2003) (e.g., Table 2 listing known antiangiogenicfactors); and Sato. Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table1 lists anti-angiogenic agents used in clinical trials).

The term “anti-cancer therapy” refers to a therapy useful in treatingcancer. Examples of anti-cancer therapeutic agents include, but are notlimited to, e.g., surgery, chemotherapeutic agents, growth inhibitoryagents, cytotoxic agents, agents used in radiation therapy,anti-angiogenesis agents, apoptotic agents, anti-tubulin agents, andother agents to treat cancer, such as anti-HER-2 antibodies (e.g.,Herceptin®), anti-CD20 antibodies, an epidermal growth factor receptor(EGFR) antagonist (e.g., a tyrosine kinase inhibitor), HER1/EGFRinhibitor (e.g., erlotinib (Tarceva®)), platelet derived growth factorinhibitors (e.g., Gleevec™ (Imatinib Mesylate)), a COX-2 inhibitor(e.g., celecoxib), interferons, cytokines, antagonists (e.g.,neutralizing antibodies) that bind to one or more of the followingtargets ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGFreceptor(s), TRAIL/Apo2, and other bioactive and organic chemicalagents, etc. Combinations thereof are also included in the invention.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g. Ar²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents, and toxins such as smallmolecule toxins or enzymatically active toxins of bacterial, fungal,plant or animal origin, including fragments and/or variants thereof.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents include, butare not limited to, alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, trietylenephosphoramide,triethiylenethiophosphoramide and trimethylolomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994));dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antiobiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin, oxaliplatin and carboplatin;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate;daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar,CPT-11) (including the treatment regimen of irinotecan with 5-FU andleucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine; combretastatin;leucovorin (LV); oxaliplatin, including the oxaliplatin treatmentregimen (FOLFOX); lapatinib (Tykerb.®); inhibitors of PKC-alpha, Raf,H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cellproliferation and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition are anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX® tamoxifen), raloxifene, droloxifene,4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, andFARESTON toremifene; aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole; andanti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; vaccines such as gene therapyvaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, andVAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor;ABARELIX® rmRH; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” as used herein refers to a compound orcomposition which inhibits growth of a cell in vitro and/or in vivo.Thus, the growth inhibitory agent can be one which significantly reducesthe percentage of cells in S phase. Examples of growth inhibitory agentsinclude agents that block cell cycle progression (at a place other thanS phase), such as agents that induce G1 arrest and M-phase arrest.Classical M-phase blockers include the vincas (vincristine andvinblastine), TAXOL®, and topo II inhibitors such as doxorubicin,epirubicin, daunorubicin, etoposide, and bleomycin. Those agents thatarrest G1 also spill over into S-phase arrest, for example, DNAalkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13.

The term “prodrug” as used in this application refers to a precursor orderivative form of a pharmaceutically active substance that is lesscytotoxic to tumor cells compared to the parent drug and is capable ofbeing enzymatically activated or converted into the more active parentform. See, e.g., Wilman, “Prodrugs in Cancer Chemotherapy” BiochemicalSociety Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) andStella et al., “Prodrugs: A Chemical Approach to Targeted DrugDelivery,” Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267,Humana Press (1985). The prodrugs described herein include, but are notlimited to, phosphate-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,D-amino acid-modified prodrugs, glycosylated prodrugs,.beta.-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs which can be converted into the more activecytotoxic free drug. Examples of cytotoxic drugs that can be derivatizedinto a prodrug form for use in this invention include, but are notlimited to, those chemotherapeutic agents described above.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone time administration and typical dosages range from 10 to 200 units(Grays) per day.

By “reduce or inhibit” is meant the ability to cause an overall decreasepreferably of 20% or greater, 30% or greater, 40% or greater, 45% orgreater, more preferably of 50% or greater, of 55% or greater, of 60% orgreater, of 65% or greater, of 70% or greater, and most preferably of75%, 80%, 85%, 90%, 95%, or greater. Reduce or inhibit can refer to, forexample, the symptoms of the disorder being treated, the presence orsize of metastases or micrometastases, the size of the primary tumor,the presence or the size of the dormant tumor, or the size or number ofthe blood vessels in angiogenic disorders.

The term “intravenous infusion” refers to introduction of a drug intothe vein of an animal or human subject over a period of time greaterthan approximately 5 minutes, preferably between approximately 30 to 90minutes, although, according to the invention, intravenous infusion isalternatively administered for 10 hours or less. The term “intravenousbolus” or “intravenous push” refers to drug administration into a veinof an animal or human such that the body receives the drug inapproximately 15 minutes or less, preferably 5 minutes or less.

The term “subcutaneous administration” refers to introduction of a drugunder the skin of an animal or human subject, preferable within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle. The pocket can be created by pinchingor drawing the skin up and away from underlying tissue.

The term “subcutaneous infusion” refers to introduction of a drug underthe skin of an animal or human subject, preferably within a pocketbetween the skin and underlying tissue, by relatively slow, sustaineddelivery from a drug receptacle for a period of time including, but notlimited to, 30 minutes or less, or 90 minutes or less. Optionally, theinfusion can be made by subcutaneous implantation of a drug deliverypump implanted under the skin of the animal or human subject, whereinthe pump delivers a predetermined amount of drug for a predeterminedperiod of time, such as 30 minutes, 90 minutes, or a time periodspanning the length of the treatment regimen.

The term “subcutaneous bolus” refers to drug administration beneath theskin of an animal or human subject, where bolus drug delivery ispreferably less than approximately 15 minutes, more preferably less than5 minutes, and most preferably less than 60 seconds. Administration ispreferably within a pocket between the skin and underlying tissue, wherethe pocket is created, for example, by pinching or drawing the skin upand away from underlying tissue.

A “disorder” is any condition that would benefit from treatment with,for example, an antibody described herein. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include cancer; benign andmalignant tumors; leukemias and lymphoid malignancies; neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders; and inflammatory, angiogenic andimmunologic disorders.

The word “label” when used herein refers to a detectable compound orcomposition which is conjugated directly or indirectly to thepolypeptide. The label can be itself be detectable (e.g., radioisotopelabels or fluorescent labels) or, in the case of an enzymatic label, cancatalyze chemical alteration of a substrate compound or compositionwhich is detectable.

By “subject” is meant a mammal, including, but not limited to, a humanor non-human mammal, such as a bovine, equine, canine, ovine, or feline.Preferably, the subject is a human. Patients are also subjects herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that DEspR is a key angiogenesis player in embryoinicdevelopment using DEspR (formerly called Dear, deposited in Gen Bank asDear) null or knockout (Dear^(−/−)) mice.

FIG. 2 shows that DEspR contributes to adult tissue vascularity as seenin adult haplo-deficient (+/−) mice exhibiting decreased tissuevascularity using power Doppler analysis.

FIGS. 3A-3E show that DEspR and VEGFsp are detected by immunostaining inumbilical vein endothelial cells (HUVECs) (FIGS. 3A-3C) andmicrovascular endothelial cells (HMECS) under both basal and angiogenictube-formation conditions. Importantly, inhibition of angiogenesisneovessel tube length is seen using both anti-DEspR (Abl) andanti-VEGFsp (Ab2) antibodies in HUVECs (FIG. 3D) and HMECs (FIG. 3E)angiogenesis assays. (Tukey's all pairwise multiple comparison P<0.001for both HUVECs and HMECs). Similar findings were observed for otherangiogenesis parameters including neovessel branching andinter-connections made.

FIGS. 4A-4D demonstrate that DEspR and VEGFsp are also detected in tumorcells, with colocalization of VEGFsp and DEspR in the cell membrane andnuclear membrane using immunostaining DEspR cell-membrane andnuclear-membrane expression are detected in multiple tumor cell types,indicating that anti-DEspR therapy is effective for different cancertypes. DEspR expression is detected in human lung non-small cell caNCI-H727, lung giant cell tumor TIB-223/GCT; breast adenoca MDA-MB-231(FIGS. 4A-4C) and MDA-MB-468, bladder ca 253J BV, colon adenoca SW480,hepatocellular ca, HEP3B, melanoma SK-MEL-2, osteosarcoma MG-63, ovarianadenoca HTB-161/NIH:OVCA R3, prostate adeno ca PC-3mm2, and pancreaticca CRL-1469/PANC-1 (FIG. 4D).

FIGS. 5A-5C show that DEspR expression was not detected in in HCI-H292lung mucoepidermoid ca, and HEPG2 hepatocellular ca (FIG. 5A), andCCL-86/Raji Burkitt's lymphoma, thus showing specificity of positiveobservations. Findings in NCI-727 lung ca cells (FIG. 5B) arecorroborated on tumor-section immunostaining of Gr.III lung adenoca(FIG. 5C).

FIGS. 6A-6B show that in contrast to control (C) and pre-immune abtreatment (PI), DEspR-inhibition via anti-human DEspR antibody treatmentinhibits tumor cell invasiveness in two cell lines tested, metastaticbreast tumor MDA-MB-231 and pancreatic adenocarcinoma PANC-1 cell lines.

FIG. 7 shows that anti-DEspR treated rats ( ) exhibited minimal tumorgrowth compared with mock-treated controls (▪), two-tailed t-test*P<0.05; **P<0.001.

FIGS. 8A-8D show, using immunohistochemical analysis, that DEspR isexpressed in mammary tumors cells (FIG. 8A) similar to MDA-MB-231 breastcancer cells, with no expression in normal breast tissue (FIG. 8B). Inaddition, residual tumors in treated rats exhibited normalization ofblood vessels (FIG. 8C) in contrast to mock-treated tumors which showeddisrupted endothelium in tumor vessels with encroachment of tumor cellsinto the lumen (FIG. 8D).

FIG. 9 shows characterization of selected monoclonal antibodies.Monoclonal antibodies 2E4A8, 2E4B11, 2E4H10, 5G12E8, 7C5B2, 7C5C5,8E7D11, 8E2F6, E2G4 and 8E7F8 were tested by indirect ELISA usingstandard procedures. Serial dilutions from supernatants containingmonoclonal antibodies at 1 μg/ml were tested as follows: 1=1/2; 2=1/4;3=1/8; 4=1/16; 5=1/32; 6=1/64; 7=1/128; 8=1/256; 9=1/512; 10=1/1024;11=1/2048 and 12=1/4096.

FIG. 10 shows Western blot analysis of monoclonal antibodies tested. Toascertain specificity, low-(5G12E8), mid-(2E4H6), and high-affinity(7C5B2) monoclonal antibodies were tested as well as the subclonesupernatant, and the subsequent purified antibody. The anti-human DEspRmonoclonal antibodies are specific for the predicted 10 kD protein forhuman DEspR. Western blot analysis was performed using total cellularprotein isolated from Cos1 human DEspR-transfected cells as antigen,primary antibody comprised purified antibody and subclone supernatant of3 selected clones, 10% gel concentration in order to detect the expected10 kD molecular weight protein of human DEspR. Nitrocellulose (PIERCE)with a transfer buffer of 3.07 g Tris, 14.4 g Glycine, 200ml methanol,800 ml dH₂0 were used. HRP-anti mouse polyvalent immunoglubulins (Sigma#0412) were used at 1:100,000; ECL reagent (SuperSignal West Femto Kit#34094), Stain reagent Kodak RP-X-Omat, and x-film (Kodak X-film#XBT-1). The Western blot results demonstrate specificity of anti-humanDEspR monoclonal antibodies regardless of relative affinity, thusidentifying more than one successful anti-human DEspR monoclonalantibody. The results indicate that the monoclonal antibody clone withhighest relative affinity and specificity is clone 7C5B2.

FIGS. 11A-11C show inhibition of different parameters of angiogenesis bymonoclonal antibody 7C5B2 and a polyclonal antibody preparation toDEspR. 7C5B2 monoclonal antibody was shown to immunostain HUVECsundergoing tube formation, pancreatic adenoca PANC-1, and breast cancerMDA-MB-231 cells. FIG. 11A shows mean number of branchpoints as ameasure of neovessel complexity, and total length of tubes as a measureof neovessel density is shown in FIG. 11B. FIG. 11C showsconcentration-dependent inhibition of in vitro serum-induced HUVECtubulogenesis by monoclonal antibody 7C5B2. HUVEC (human umbilical veinendothelial cells) were grown onto Matrigel-coated wells in basal mediumsupplemented with 2% FBS (control), or 2% FBS+monoclonal antibody 7C5B2(0.05-500 nM). The percentage of serum-induced tubulogenesis wasdetermined as the difference between HUVECs grown in control conditionsand the indicated monoclonal antibody 7C5B2-supplemented media. The % ofthe total tube length per well and the total number of branching pointsper well in the in vitro tube formation assay is presented. Data areshown as mean±standard error. Each experimental condition was performedin five replica wells. EC₅₀ for total tube length=4.34±0.45 nM; EC₅₀ for# branching points=3.97±0.51 nM.

FIGS. 12A-12C demonstrate that a monoclonal antibody 7C5B2 inhibitstumor cell invasiveness in MDA-MB-231 human breast cancer (FIG. 12A) andPANC-1 pancreatic cancer (FIG. 12B) cell lines (P<0.001*, <0.01*). FIG.12C shows dose response curve of inhibition of MDA-MB-231 cell invasionby monoclonal antibody 7C5B2 (EC₅₀=3.55±0.32 nM). Data, mean±standarderror of 5 replicates. *P<0.001, **P<0.01 (one way ANOVA, all pairwisemultiple comparison Tukey's Test).

FIGS. 13A-13D show effects of an anti-human DEspR monoclonal antibody7C5B2 (IgG2b isotype) on in vitro serum-induced HUVEC tubulogenesis(established in vitro angiogenesis assay). HUVECs (human umbilical veinendothelial cells) were grown onto Matrigel-coated wells in basal mediumsupplemented with 2% FBS (control C1), or 2% FBS+pre-immune IgG isotypecontrol for polyclonal anti-hDESPR antibody (500 nM, control C2), or 2%FBS+IgG2b isotype control for anti-hDESPR mAB (500 nM, C3 control) or 2%FBS+polyclonal anti-hDEspR (500 nM, P) or 2% FBS+monoclonal antibody7C5B2 (500 nM, M). Quantitative analysis of the mean number of tubesformed per well is shown in FIG. 13A, the mean number of branchingpoints per well is shown in FIG. 13B, the mean number of connections perwell is shown in FIG. 13C and the mean total tube length in mm per wellis shown in FIG. 13D, using the in vitro tube formation assay. Data areshown as mean±standard error. Each experimental condition was performedin five replica wells. Statistically significant differences (ascompared with respective control conditions), are indicated as follows:*P<0.001 (one way ANOVA followed by all pairwise multiple comparisonTukey Test).

FIGS. 14A-14B show immunohistochemical analysis of human tumortissue-arrays comprised of core biopsy specimens representing tumors andnormal tissue on the same slide using an anti-human DEspR 7C5B2monoclonal antibody. Conditions that optimized specificity andsensitivity of detection were first tested using formalin-fixed,paraffin embedded core biopsy sections. Double-immunofluorescenceexperiments were performed to evaluate human DEspR expression and CD133expression, with the latter serving as a marker for cancer stem cells.Antigen-retrieval was performed using anti-human DEspR monoclonalantibody at 1:10, and commercially available anti-CD133 monoclonalantibody at 1:20 dilutions. Representative immunohistochemical analysisof human tumor tissue-arrays using anti-human DEspR 7C5B2 monoclonalantibody detected increased expression of hDEspR (Alexa-568red) in stageII-lung cancer tumor cells, as shown in FIG. 14A. Some tumor cells wereimmunostained double -positive for both human DEspR and CD133, whileother tumor cells immunostained only for CD133. These observationsdemonstrate that human DEspR is also present in CD133-positive cancerstem cells, as well as CD133-negative tumor cells. As shown in FIG. 14B,in contrast, a normal lung specimen does not exhibit any immunostainingfor human DEspR or CD133.

FIGS. 15A-15B show that there is minimal DEspR expression in normalhuman pancreas (FIG. 15B), with α-smooth muscle actin serving aspositive control, while, in contrast, stage IV pancreatic cancer tumorcells and tumor blood vessels exhibits increased DEspR expression (FIG.15A).

FIGS. 16A-16D demonstrate DEspR-targeted ultrasound molecular imagingand show that a DEspR-specific antibody (FIG. 16A) detectsDEspR+endothelial lesions (FIG. 16B) and vasa vasorum angiogenesis (FIG.16C). Quantitation of contrast intensity is done using integratedVisualSonics Micro-imaging System software (FIG. 16D) and demonstratesincreased contrast intensity in DEspR+carotid artery endothelium andvasa vasorum, in contrast to both low contrast intensity in DEspR(−)endothelium and vasa vasorum, and isotype-microbubble controls.P<0.0001, ANOVA and pairwise multiple comparison. Anti-DEspR-antibody isbiotinylated and coupled to streptavidin-PEG coated commerciallyavailable microbubbles for ultrasound analysis and imaging.

FIGS. 17A-17F show immunohistochemical analysis of DEspR expression inhuman breast tissue using an anti-DEspR monoclonal antibody (FIGS.17A-17C) normal; Grade-1, T1 invasive ductal carcinoma (FIGS. 17D-17F).FIG. 17A shows normal breast tissue: 3×-overlay of DEspR, aSMA and DAPInuclear stain detects aSMA expression in mammary myoepithelial cells butno expression of DEspR in epithelial cells and microvessels. FIG. 17Bshows 2×-immunofluorescence overlay of DEspR and DAPI nuclear stain andconfirms absence of DEspR expression in normal breast tissue. FIG. 17Cis a 4×-overlay of DEspR, aSMA, DAPI immunofluorescence and diffusioncontrast imaging (DIC) that delineates tissue morphology, expression ofaSMA. and non/minimal-expression of DEspR in normal mammary epitheliumand endothelium. FIG. 17D is a 3×-Overlay of DAPI, aSMA and DEspRimmunofluorescence in Gr.I-T1 invasive ductal carcinoma that detectsDEspR expression in vascular endothelium, and co-localization with aSMAin mammary tissue. FIG. 17E is a 2×-overlay of DAPI and DEspR of breastcancer shown in panel 17D that highlights DEspR expression. FIG. 17F isa 4×-overlay of DAPI, aSMA, DEspR, DIC to elucidate DEspR spatialexpression with tissue morphology of epithelial cells and microvessels.bar=20 microns.

FIGS. 18A-18F show monoclonal antibody immunohistochemical analysis ofDEspR expression in normal pancreatic tissue (FIGS. 18A-18C) normal; andGrade-3, T3 pancreatic ductal carcinoma (FIGS. 18D-18F). FIG. 18A showsthat normal pancreatic tissue, with a 3×-overlay of DEspR, aSMA and DAPInuclear stain, detects minimal DEspR expression in microvessels. FIG.18B shows a 4×-immunofluorescence overlay of DEspR, aSMA, DAPI, with DICimaging of tissue morphology. FIG. 18C (left) shows a 3×-overlay ofDEspR, aSMA, DAPI immunofluorescence; (right) shows a 4×-overlay ofDEspR, aSMA, DAPI and diffusion contrast imaging (DIC) for tissuemorphology that shows aSMA expression and non/minimal-expression ofDEspR in normal endothelium. FIG. 18D shows that 3×-overlay of DAPI,aSMA and DEspR immunofluorescence in Gr.3-T3 pancreatic ductal carcinomadetects DEspR expression in vascular endothelium, and co-localizationwith aSMA. FIG. 18E shows a 2×-overlay of DAPI and DEspR of the imageshown in FIG. 19D and highlights DEspR expression. FIG. 18F shows a3×-overlay of DAPI, aSMA, DEspR, that shows increased DEspR expressionin pancreatic ductal carcinoma cells. bar=20 microns.

FIGS. 19A-19E show representative contrast enhanced ultrasound(CEU)-images with contrast intensity signals (CIS) depicted. FIG. 19Ashows MB_(D) DEspR-targeted molecular imaging in transgenic rat-R1demonstrating CEU-positive imaging and the characteristic drop inCIS-peak after acoustic disruption (|). FIG. 19B shows subsequentisotype-microbubble (MB_(C)) imaging in transgenic rat-R1 showing lowpeak CIS-levels and ‘flat-line pattern of CIS pre- and post-destructionindicating CIU-negative imaging. FIG. 19C shows MB_(D) DEspR-targetedmolecular imaging in non-transgenic rat-R2 demonstrating CEU-negativeimaging similar to MB_(C) CEU-negative imaging. FIG. 19D shows a graphof CIS-differences (Δ) among different study groups as notateddistinguishing CEU-positive imaging in Tg MB_(D) CEU+ group from theother CEU-negative groups. FIG. 20E shows a graph of CIS-differencebetween all transgenic rats (Tg+) and non-transgenic rats (nonTg).Hatched bar represents a threshold between MB_(D)-infused CEU+ andMB_(D)-infused CEU-transgenic rats. Blood pool, CEU-image 1 minute afterbolus injection of MBs, demonstrating equivalent MB-infusion amongdifferent rats and minimal contrast-intensity signals from movementartifacts. 1-Pre, pre-acoustic destruction CEU-images obtained 4-minutesafter bolus infusion, in order to allow MB-adherence to target, if any,and to document minimal, if any, circulating MBs in the lumen. Imagecorresponds to #1 on CIS-plot. 2-Post, CEU-image after acousticdestruction corresponding to #2 on scatter plot. CIS-plot, scatter plotof contrast-intensity signals (CIS) in representative regions ofinterest (encircled in aqua). #1, CIS detected pre-acoustic destruction;#2, CIS detected post-acoustic destruction (2). Black line and followinggap mark period of acoustic destruction in CIS-scatter plots. MB_(D),DEspR-targeted microbubble; MB_(C), control isotype-targetedmicrobubble; Tg, transgenic rat; nonTg, nontransgenic control rat; CEU+,CEU positive imaging; CEU−, CEU negative imaging, Δ Contrast Intensity,pre-/post-destruction CIS-difference; ***, P<0.0001.

FIGS. 20A-20H depict representative MB_(D)-specific contrast enhancedultrasound (CEU)-positive images depicting complex pattern of acousticdestruction of adherent MB_(D)-microbubbles in a transgenic rat, R3.FIG. 20A shows representative CEU-image documenting blood pool ofcirculating MB_(D)s filling carotid artery lumen one-minute after bolusinfusion. CCA, common carotid artery; ECA, external carotid artery; ICA,internal carotid artery; *, CCA bifurcation. FIGS. 20B-20D show scatterplots of contrast-intensity signals marked with same-dashed blocks torefer to corresponding regions of interest (ROI) in panel-20E. (20B)white solid line; (20C), white hatched line; (20D) white dotted lineROIs. FIG. 20E shows representative CEU-image that corresponds to #1 onscatter plots b,c,d documenting adherent DEspR-targeted microbubbles(MB_(D)) just prior to pre-acoustic destruction (black line). AdherentMB_(D)s are seen in the three ROIs encircled white solid line, whitehatched line, and white dotted line. FIG. 20F shows representativeCEU-image corresponding to #2 on scatter plots b-d showing apost-acoustic destruction dip in signal intensity compared to levels in#1 in the different ROIs respectively. FIG. 20G shows representativeCEU-image corresponding to #3 on scatter plots b-d showing apost-acoustic destruction secondary peak in contrast intensity signalsin the different ROIs. FIG. 20H shows representative CEU-imagecorresponding to #4 on scatter plots documenting the decline incontrast-intensity signals approaching baseline levels observed inisotype control or MB_(D)-infused CEU-negative images and demonstratinglow background CIS levels.

FIGS. 21A-21H depict representative histological and fluorescenceimmunostaining analysis of carotid arteries with DEspR-positivemolecular imaging corresponding to rat-R1 (panels 21A-21D), and rat-R3in (panels 21E-21H). FIG. 21A shows Masson trichrome stained section ofcarotid artery endothelium. FIGS. 21B-212C show differentialinterference contrast (DIC) image overlaid with fluorescenceimmunostaining for DEspR expression and DAPI nuclear stain. FIG. 21Dshows control isotype-ab immunostaining and DAPI nuclear stain overlaidwith DIC image of endothelium. FIG. 21E shows carotid artery Massontrichrome-stained section showing increased adventitial vasa vasorumneovessels. Boxed area is shown in higher magnification in FIG. 21Fdocumenting rbc-filled vasa vasorum. FIG. 21G shows fluorescenceimmunostaining detects DEspR-positive expression in vasa vasorum andsurrounding cells. FIG. 21H shows double immunostaining with α-SMA andDEspR detects αSMA co-expression in DEspR-positive neovessels. →,adherent DEspR-targeted microbubble MB_(D); white arrowheads point tovasa vasorum neovessel in panels 21G and 21H; m, media; bar=10-micronspanels 21A-21D, 21F; 20 microns panels 21E, 21G, 21H.

FIGS. 22A-22E depict representative fluorescence immunostaining analysisof carotid arteries from rats exhibiting MB_(D)-specific CEU-positiveimaging (22B, 22C) and CEU-negative imaging (22D, 22E). FIG. 22A showsscatter dot plot of pre-destruction CIS-peak levels highlighting athreshold (hatched bar) between MB_(D)-specific CEU-positive (CEU+) andCEU-negative (CEU−) imaging. FIG. 22B shows DEspR-positiveimmunostaining of carotid artery endothelium and expanded vasa vasorum;αSMA-positive immunostaining in smooth muscle cells (SMCs) in the media.Some vasa vasorum neovessels are double-immunostained for DEspR andαSMA. FIG. 22C shows corresponding DIC-image shows structural layers ofcarotid artery and vasa vasorum. FIG. 22D shows representative minimalto no DEspR-expression in rat carotid artery exhibiting CEU-negativeimaging (shown here, nonTg rat-R2). Similar images obtained forCEU-negative transgenic rat carotid arteries. αSMA-immunostainingdetects expression in SMCs in the media. Low levels ofαSMA-immunostaining in the medial indicates synthetic SMC phenotype inboth carotid arteries, consistent with hypertensive remodeling. FIG. 22Dshows corresponding DIC-image shows structural layers of carotid arteryand adventitia with no vasa vasorum expansion. Bar=20 microns (22B,22C), 10 microns (22D, 22E). m, media; adv, adventitia; white smallarrow, endothelium; white large arrow, vasa vasorum.

FIGS. 23A-23G depicts phase contrast-fluorescence microscopy analysis ofanti-humanDEspR-targeted microbubbles (MB_(D)) binding to humanendothelial cells, HUVECs, in vitro. Increasing DEspR-targetedmicrobubbles (MB_(D)) to cell ratio (23A) 8×, (23B) 80×, and (23C) 800×.(23D) Isotype control (MB_(C)) at 800×; (23E) non-targeted controlMB_(O) at 800×. (23F) % of HUVECs with bound MBs (▪) and no MB binding(□). FIG. 23G shows number of MBs (mean +/−sem) per bound cell withincreasing MB to cell ratio: MB_(D) compared with isotype control MB_(C)and control non-targeted MB_(O). ***, ANOVA P<0.0001.

FIGS. 24A-24F show DEspR expression in liver (24A-24C) and pancreas(24D-24F) non-cancerous and cancerous tissues. (24A) Adjacent normalliver tissue; (24B, 24C) hepatic carcinoma T-2 from two patients; (24D),adjacent normal pancreatic tissue; (24E, 24F) pancreatic ductalcarcinoma Grade III-IV from two patients. Black arrow, microvessels;DAB-detection of DEspR-positive immunostaining with color-intensityroughly proportional to expression; hematoxylin nuclear counterstain.Bar, 20 microns.

FIGS. 25A-25F show DEspR expression in a human tissue array: stomach(25A-25C) and breast (25D-25F) non-cancerous and cancerous tissues.(25A) Adjacent normal stomach tissue; (25B) stomach adenocarcinoma T-3,(25C) stomach adenocarcinoma metastasis to lung; (25D) adjacent normalbreast tissue with fibrosis; (25E) breast medullary carcinoma T-2; (25F)breast tumor metastasis to lymphnode. Black arrow, vascular endothelium;DAB-detection of DEspR-positive immunostaining with color-intensityroughly proportional to expression; hematoxylin nuclear counterstain.Bar, 20 microns.

FIGS. 26A-26F show DEspR expression in lung and colon non-cancerous andcancerous tissue. (26A) adjacent normal lung; (26B) Gr-I lungadenocarcinoma; (26C), Gr.III,T2 lung adenocarcinoma; (26D) adjacentnormal colon; (26E, 26F) colon adenocarcinoma Gr.III-IV, T2. white arrow, endothelium; black arrow →, DEspR-immunostaining of nuclear membranein cancer cells DAB-detection of DEspR-immunostaining withcolor-intensity roughly proportional to expression; hematoxylin nuclearcounterstain. Bar, 20 microns 26A-26C; 25 microns 26D; 10 microns 26E,26F.

FIGS. 27A-27F show DEspR expression in different tissue-type cancer celllines. (27A) non-small cell lung cancer cell line, #NCI-H727; (27B)colon carcinoma, SW480 Duke's type B; (27C) pancreatic carcinoma,PANC-1; (27D) breast adenocarcinoma metastasis, MDA-MB-231; (27E)bladder carcinoma 253J BV; (27F) prostate adenocarcinoma PC-3mm2. →,DEspR-immunostaining of nuclear membrane in cancer cells, DAB-detectionof DEspR-immunostaining with color-intensity roughly proportional toexpression; hematoxylin nuclear counterstain. Bar, 20 microns A-F.

FIGS. 28A-28B show characterization of a human-specific anti-DEspRmonoclonal antibody. (28A) Analysis by indirect ELISA of 10 candidatemonoclonal antibody clones is shown. Serial dilutions from supernatantscontaining mAbs at 1 μg/ml were tested as follows: 1=1/2; 2 =1/4; 3=1/8;4=1/16; 5=1/32; 6=1/64; 7=1/128; 8=1/256; 9=1/512; 10=1/1024; 11=1/2048and 12=1/4096. white diamond, selected Mab 7c5b2 clone, open symbols,all others. (28B) Western blot analysis of purified Mabs (lanes 1-3),and “super clone” supernatants (lanes 4-6), with PBS serving as control(lane 7) are depicted. Selected 7C5B2 Mab in lanes 1 and 4 (diamond).Double immunostaining of HUVECs with anti-DEspR Mab-immunostaining andanti-VEGFsp immunostaining was performed and colocalization of DEspR andVEGFsp determined.

FIGS. 29A-29C demonstrate that DEspR inhibition via monoclonal antibodydecreases angiogenesis in in vitro HUVECs assay. DEspR immunostaining ofHUVECs using anti-DEspR Mab was performed. (29A) Dose response curve toanti-DEspR Mab inhibition of angiogenesis measuring total tube lengthper well (◯) with EC₅₀=4.34 +/−0.45 nM; and number of tube branch points(●) with EC.₅₀3.97 +/−0.51 nM. (29B) Analysis of total tube lengthchanges upon DEspR-inhibition via anti-DEspR polyclonal (Pab) andmonoclonal (Mab) antibodies compared to control untreated cells (30C),pre-immune serum (PI) and IgG2b isotype (Iso) controls for Pab and Mab,respectively. (29C) Analysis of mean number (#) of branchpointsinhibited by Pab and Mab anti-DEspR ab-inhibition compared with controls(C, PI, Iso). Data expressed as mean +/−sem; 4 replicates; *, P<0.01(ANOVA followed by all pairwise multiple comparison Tukey test).

FIGS. 30A-30C demonstrate that DEspR inhibition via monoclonal antibodydecreases angiogenesis in in vitro HUVECs assay. DEspR-positiveimmunostaining of MDA-MB-231 breast cancer cells and PANC-1 pancreaticcancer cell line via anti-DEspR Mab was performed. (30A) Dose responsecurve to increasing DEspR-inhibition via anti-DEspR Mab of MDA=MB-231breast cancer cell invasiveness (black), EC₅₀=3.55+/−0.32 nM. (30B-30C)Analysis of cell invasiveness inhibited by anti-DEspR Mab inhibitioncompared to control untreated cells, and IgG2b isotype control forMDA-MB-231 breast cancer cells (31B), and PANC-1 pancreatic cell line(31C). All data shown as mean+/−sem of 4 replicates; *, P<0.01; **,P<0.001 (1-way ANOVA followed by all pairwise multiple comparison TukeyTest).

FIGS. 31A-31F show immunohistochemical analysis of DEspR expression inhuman breast tissue. (31A-31C) normal; (31D-31F) Grade-1, T1 invasiveductal carcinoma. 31A. Normal breast tissue: 3×-overlay of DEspR, aSMAand DAPI nuclear stain detects aSMA expression in mammary myoepithelialcells but no expression of DEspR in epithelial cells (white triangulararrowhead →) and microvessels (white rounded arrowhead). 31B,2×-immunofluorescence overlay of DEspR and DAPI nuclear stain confirmsabsence of DEspR expression in normal breast tissue. 31C, 4×-overlay ofDEspR, aSMA, DAPI immunofluorescence and diffusion contrast imaging(DIC) delineates tissue morphology, expression of aSMA. andnon/minimal-expression of DEspR in normal mammary epithelium andendothelium. 31D, 3×-Overlay of DAPI, aSMA and DEspR immunofluorescencein Gr.I-T1 invasive ductal carcinoma detects DEspR expression invascular endothelium, and co-localization with aSMA in mammary tissue.31E, 2×-overlay of DAPI and DEspR of breast cancer shown in panel dhighlights DEspR expression. 31F, 4×-overlay of DAPI, aSMA, DEspR, DICelucidate DEspR spatial expression with tissue morphology. (whitetriangular arrowhead →), epithelial cells; (white rounded arrowhead),microvessels. DEspR-positive; aSMA-positive; DAPI nuclear stain;colocalization of aSMA and DEspR; bar=20 microns.

FIGS. 32A-32F demonstrate immunohistochemical analysis of DEspRexpression in pancreatic tissue using anti-DEspR Mab. (32A-32C) normal;(32D-32F) Grade-3, T3 pancreatic ductal carcinoma. (32A) Normalpancreatic tissue: 3×-overlay of DEspR, aSMA and DAPI nuclear staindetects minimal DEspR expression in microvessels seen better in panel(32B) 4×-immunofluorescence overlay: DEspR, aSMA, DAPI, with DIC imagingof tissue morphology. (32C) left: 3×-overlay of DEspR, aSMA, DAPIimmunofluorescence; right: 4×-overlay of DEspR, aSMA, DAPI and diffusioncontrast imaging (DIC) for tissue morphology shows aSMA expression andnon/minimal-expression of DEspR in normal endothelium. (32D) 3×-overlayof DAPI, aSMA and DEspR immunofluorescence in Gr.3-T3 pancreatic ductalcarcinoma detects DEspR expression in vascular endothelium, andco-localization with aSMA. (32E) 2×-overlay of DAPI and DEspR of imageshown in panel 32D highlights DEspR expression. (32F) 3×-overlay ofDAPI, aSMA, DEspR, shows increased DEspR expression in pancreatic ductalcarcinoma cells. (white →), epithelial cells; (white rounded arrowhead,microvessels. DEspR-positive; aSMA-positive; DAPI nuclear stain;colocalization of aSMA and DEspR; bar=20 microns.

FIG. 33 demonstrates 1% agarose gel separation of RT-PCR products ofantibody obtained from the 7C5B2 hybridoma. Gel was stained with SYBR®Safe DNA gel stain (Invitrogen cat. no. 533102) and photographed overultraviolet light. Size marker (L) is GeneRuler™ 1Kb Plus (Fermentascat. no. SM1331). RT-PCR was performed using degenerate primer pools formurine signal sequences with constant region primers for each of IgGVH,IgMVH, IgκVL and IgλVL.

FIG. 34 shows the variable heavy chain amino acid (SEQ ID NO: 4) andnucleotide (SEQ ID NO: 3) sequence of the 7C5B2 antibody. CDRdefinitions and protein sequence numbering according to Kabat.

FIG. 35 shows the variable light chain amino acid (SEQ ID NO: 9) andnucleotide (SEQ ID NO: 8) sequence of a composite 7C5B2 antibody. CDRdefinitions and protein sequence numbering according to Kabat.

FIG. 36 shows an exemplary variable heavy chain amino acid (SEQ ID NO:13) and nucleotide (SEQ ID NO: 21) sequence of a composite anti-DEspRhumanized 7C5B2 antibody generated using the methods described herein.CDR definitions and protein sequence numbering according to Kabat.

FIG. 37 shows an exemplary variable heavy chain amino acid (SEQ ID NO:14) and nucleotide (SEQ ID NO: 22) sequence of a composite anti-DEspRhumanized 7C5B2 antibody generated using the methods described herein.CDR definitions and protein sequence numbering according to Kabat.

FIG. 38 shows an exemplary variable heavy chain amino acid (SEQ ID NO:15) and nucleotide (SEQ ID NO: 23) sequence of a composite anti-DEspRhumanized 7C5B2 antibody generated using the methods described herein.CDR definitions and protein sequence numbering according to Kabat.

FIG. 39 shows an exemplary variable heavy chain amino acid (SEQ ID NO:16) and nucleotide (SEQ ID NO: 24) sequence of a composite anti-DEspRhumanized 7C5B2 antibody generated using the methods described herein.CDR definitions and protein sequence numbering according to Kabat.

FIG. 40 shows an exemplary variable heavy chain amino acid (SEQ ID NO:17) and nucleotide (SEQ ID NO: 25) sequence of a composite anti-DEspRhumanized 7C5B2 antibody generated using the methods described herein.CDR definitions and protein sequence numbering according to Kabat.

FIG. 41 shows an exemplary variable light chain amino acid (SEQ ID NO:18) and nucleotide (SEQ ID NO: 26) sequence of a composite anti-DEspRhumanized 7C5B2 antibody generated using the methods described herein.CDR definitions and protein sequence numbering according to Kabat.

FIG. 42 shows an exemplary variable light chain amino acid (SEQ ID NO:19) and nucleotide (SEQ ID NO: 27) sequence of a composite anti-DEspRhumanized 7C5B2 antibody generated using the methods described herein.CDR definitions and protein sequence numbering according to Kabat.

DETAILED DESCRIPTION

Provided herein are novel compositions comprising anti-DEspR antibodiesand DEspR-binding fragments thereof, and methods of their use inanti-angiogenesis and anti-tumor proliferation and invasivenesstherapies, such as the treatment of cancer, as well as the treatment ofthose vascular diseases where pathological angiogenesis plays a role,such as in carotid artery disease, vasa vasorum neovascularization (thusimpacting, for example, stroke), and vulnerable plaqueneovascularization (thus impacting, for example, heart disease). Inaddition, the compositions comprising the anti-DEspR antibodies andDEspR-binding fragments thereof described herein are useful inassessment and imaging methods, such as companion diagnostics fordetermining DEspR expression in tumor biopsies to identify likelyreponders for personalized medicine approaches, DEspR-targeted molecularimaging of angiogenesis, which can be used, for example, in serialmonitoring of response(s) to therapy, in vivo detection of tumor“angiogenic switch,” or vascular mimicry. Further, such diagnosticsprovide novel approaches for anti-angiogenic therapies for use inpersonalized medicine applications. Furthermore, the compositionscomprising the anti-DEspR antibodies and DEspR-binding fragments thereofdescribed herein are useful as targeting moieties for other diagnosticand therapeutic compositions, in combination with delivery agents suchas nanoparticles, polyplexes, microparticles, etc.

Therapies targeting the VEGF and VEGFR2 receptor pathways, such asbevacizumab, sunitinib, and sorafanib treatments, have recently beenshown to have only transitory benefits, and appear to promote or inducea feedback angiogenic response, such that 10-fold increases in VEGFlevels have been detected following anti-VEGF treatment (Willett et al.,2005 and Carmelie et al. 2005), and exacerbations of metastasis havebeen seen after VEGFR2 inhibitor (sunitinib) treatments.

In contrast, the inventors have discovered another angiogenesis arm ofthe VEGF system, based on their discovery that key, non-redundant, anddistinct roles are played by interaction of the VEGF signal peptide(VEGFsp) with its receptor “DEspR” or “dual endothelin-1/VEGFspreceptor”. The inventors have found that: a) a DEspR null mutation leadsto E10.5-E12.5 embryonic lethality due to abnormal embryonicvasculogenesis and angiogenesis (Herrera et al. 2005); b) VEGFsp bindsDEspR with high affinity, equal to what is observed for ET1 binding(Herrera et al. 2005); c) DEspR antibody-mediated inhibition in a ratmammary tumor model and DEspR haplo-deficiency in DEspR+/−mice reducestumor growth in vivo (Herrera et al. 2005); d) VEGFsp stimulates adultrat aortic ring angiogenesis (Decano et al., 2010); and e) DEspRmediates adult angiogenesis and its expression is increased duringcarotid atherosclerotic vasa vasorum neovascularization, as describedherein.

As described herein, the inventors further demonstrate that: a) DEspRexpression is increased in several human cancer tumor vessels in bothmales and females (e.g., breast, lung, liver, bladder, pancrease,stomach, esophagus, colon, etc.), and surprisingly, also in a variety oftumor cells, including breast, lung, glioblastoma, bladder, melanoma,and pancreatic tumor cells, using tumor tissue arrays and tumorcell-line arrays respectively; as well as in cancer stem cells or cancerstem-like cells or tumor initiating cells; b) DEspR and VEGFsp arecolocalized in both nuclear and cell membranes in cultured tumor cells;c) VEGFsp stimulates both tumor cell proliferation and invasiveness; andthat d) DEspR inhibition via polyclonal and monoclonal anti-human DEspRantibodies potently suppress angiogenesis and tumor cell invasiveness,and reduce tumor growth rate and decrease tumor size significantly.

DEspR

The dual endothelin-1NEGF signal peptide activated receptor (DEspR),formerly DEAR was originally cloned from a Dahl salt-sensitivehypertensive rat brain cDNA library and was shown to be a singletransmembrane receptor coupled to a Ca2+-mobilizing transduction pathwaybinding endothelin-1 (ET-1) and angiotensin-II (Ang II) with equivalentaffinities (Ruiz-Opazo N. et al. (1998), Molecular characterization of adual Endothelin-1/Angiotensin II Receptor. Mol Med. 4: 96-108).Subsequent molecular studies elucidated that the mouse ortholog does notinteract with AngII but binds ET-1 and the vascular endothelial growthfactor signal peptide (VEGFsp) with equal affinities instead.DEspR^(−/−) double mutant deficiency in mice resulted in embryoniclethality due to impaired vasculogenesis, abnormal angiogenesis andvascular network formation. DEspR^(−/−) embryos also showed abnormalneurogenesis marked by a hyperconvoluted neuroepithelium anddysregulated neural tube differentiation from the telencephalon tomyelencephalon (Herrera VLM, et al., (2005), Embryonic lethality in Deargene deficient mice: new player in angiogenesis. Physiol. Genomics 23:257-268.). This phenotype is strikingly opposite to the proapoptoticeffects observed in the developing neural tube in VEGF^(+/−) deficientmice, although abnormalities in vasculogenesis and angiogenesis aresimilar (Herrera VLM, et al., (2005)).

Accordingly, the term “ DEspR,” as used herein, refers to the 85-aminoacid dual endothelin/VEGF signal peptide receptor (DEspR) having thehuman amino acid native sequence of:MTMFKGSNEMKSRWNWGSITCIICFTCVGSQLSMSSSKASNFSGPLQLYQRELEIFIVLTDVPNYRLIKENSHLHTTIVDQGRTV (SEQ ID NO:1), as described by, e.g., Glorioso etal. 2007, together with naturally occurring allelic, splice variants,and processed forms thereof.

As used herein a DEspR “native sequence” or DEspR “wild-type sequence”polypeptide comprises a polypeptide having the same amino acid sequenceas a DEspR polypeptide derived from nature. Thus, a native sequencepolypeptide can have the amino acid sequence of naturally-occurringpolypeptide from any mammal. Such native sequence polypeptide can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence” polypeptide specifically encompassesnaturally-occurring truncated or secreted forms of the polypeptide(e.g., an extracellular domain sequence), naturally-occurring variantforms (e.g., alternatively spliced forms) and naturally-occurringallelic variants of the polypeptide.

A DEspR polypeptide “variant” means a biologically active DEspRpolypeptide having at least about 80% amino acid sequence identity witha native sequence of a DEspR polypeptide. Such variants include, forinstance, polypeptides wherein one or more amino acid residues areadded, or deleted, at the N- or C-terminus of the polypeptide.Ordinarily, a variant has at least about 80% amino acid sequenceidentity, more preferably at least about 90% amino acid sequenceidentity, and even more preferably at least about 95% amino acidsequence identity with the native sequence polypeptide.

DEspR is part of the G protein coupled receptor family, and binds toendothelin-1 and to VEGF signal peptide (VEGFsp). VEGFsp has the humansequence MNFLLSWVHWSLALLLYLHHAKWSQA (SEQ ID NO:2). Typically, as usedherein, DEspR refers to human DEspR. The term “DEspR” is also used torefer to truncated forms or fragments of the polypeptide comprisingspecific amino acids sequences of the 85-amino acid human dualendothelin/VEGF signal peptide receptor. Reference to any such forms ofDEspR can be identified in the application, e.g., by “DEspR (1-9).”

DEspR Antagonists & Anti DEspR Antibodies

Provided herein are compositions and methods comprising DEspRantagonists that are capable of neutralizing, blocking, inhibiting,abrogating, reducing, or interfering with DEspR activities including itsbinding to endothelin-1 or VEGFsp. DEspR antagonists include, but arenot limited to, anti-DEspR antibodies and antigen-binding fragmentsthereof, receptor molecules, small molecules, nanoparticles, polyplexcombinations and derivatives thereof that bind specifically to DEspRthereby inhibiting, preventing, or sequestering its binding to itsligands, such as VEGFsp and endothelin-1.

Anti-DEspR Antibodies and Antibody Production

Accordingly, in some aspects, provided herein is an anti-DEspR antibodyor antibody fragment thereof that is specific for a DEspR target, wherethe anti-DEspR antibody or antibody fragment thereof specifically bindsto the DEspR target and reduces or inhibits DEspR biological activity.In some embodiments, the DEspR is human DEspR. In some embodiments, theDEspR target comprises an amino acid sequence of SEQ ID NO:1 or anallelic or splice variant thereof.

As used herein, an “anti-DEspR antibody” refers to an antibody thatbinds to DEspR with sufficient affinity and specificity. The antibodyselected will normally have a binding affinity for DEspR, for example,the antibody can bind human DEspR with a K_(D) value between 10⁻⁵ M to10⁻¹⁰ M. As used herein, “selectively binds” or “specifically binds”refers to the ability of an anti-DEspR antibody or antibody fragmentthereof described herein to bind to DEspR, with a K_(D) 10⁻⁵ M (10000nM) or less, e.g., 10⁻⁶ M, 10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M,10⁻¹² M, or less.

Antibody affinities can be determined, for example, by a surface plasmonresonance based assay (such as the BIAcore assay described in PCTApplication Publication No. WO2005/012359); enzyme-linkedimmunoabsorbent assay (ELISA); and competition assays (e.g. RIA's), forexample. In certain aspects described herein, an anti-DEspR antibody canbe used as a therapeutic agent in targeting and interfering withdiseases or conditions where DEspR activity is involved. Also, theanti-DEspR antibody can be subjected to other biological activityassays, e.g., in order to evaluate its effectiveness as a therapeutic,or its effectiveness as a diagnostic aid, etc. Such assays are known inthe art and depend on the target antigen and intended use for theantibody. Examples include the HUVEC inhibition assay; tumor cell growthinhibition assays (as described in WO 89/06692, for example);antibody-dependent cellular cytotoxicity (ADCC) and complement-mediatedcytotoxicity (CDC) assays (U.S. Pat. No. 5,500,362); and agonisticactivity or hematopoiesis assays (see WO 95/27062). Other biologicalactivity assays that can be used to assess an anti-DEspR antibody aredescribed herein in the Examples section.

Thus, anti-DEspR antibodies or antibody fragments thereof that areuseful in the compositions and methods described herein include anyantibodies or antibody fragments thereof that bind with sufficientaffinity and specificity to DEspR, i.e., are specific for DEspR, and canreduce or inhibit the biological activity of DEspR.

Accordingly, in some aspects, provided herein is an anti-DEspR antibodyor antibody fragment thereof that binds to DEspR and inhibits DEspRbiological activity or blocks interaction of DEspR with VEGFsp. In someembodiments of these aspects and all such aspects described herein, theVEGFsp has a sequence comprising the sequence of SEQ ID NO: 2. In someembodiments of these aspects and all such aspects described herein, theanto-DEspR antibody or antibody fragment thereof is specific for anepitope of DEspR comprising an extracellular portion of DEspR. In someembodiments of these aspects and all such aspects described herein, theanti-DEspR antibody or antibody fragment thereof is specific for anepitope of DEspR comprising amino acids 1-9 of SEQ ID NO: 1.

Further description and examples of anti-DEspR antibodies and antibodyfragments thereof useful with the compositions and methods describedherein, as well as methods of making and characterizing the same, areprovided below:

Polyclonal Antibodies

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen, e.g., DEspR(1-9) and an adjuvant. It can be useful, in someembodiments, to conjugate the relevant antigen to a protein that isimmunogenic in the species to be immunized, e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsininhibitor using a bifunctional or derivatizing agent, for example,maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteineresidues), N-hydroxysuccinimide (through lysine residues),glutaraldehyde, succinic anhydride, SOC1₂, or R¹N=C=NR, where R and R¹are different alkyl groups.

Animals can be immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with ⅕ to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer Animals areboosted until the titer plateaus. Preferably, the animal is boosted withthe conjugate of the same antigen, but conjugated to a different proteinand/or through a different cross-linking reagent. Conjugates also can bemade in recombinant cell culture as protein fusions. Also, aggregatingagents such as alum are suitably used to enhance the immune response.

Monoclonal Antibodies

Preferably, anti-DEspR antibodies or antibody fragments thereof for usewith the compositions and methods described herein are anti-DEspRmonoclonal antibodies or fragments thereof. The term “monoclonalantibody” refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that can be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a single antigen.Furthermore, in contrast to polyclonal antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. Various methods for making monoclonalantibodies specific for DEspR as described herein are available in theart. For example, the monoclonal antibodies can be made using thehybridoma method first described by Kohler et al., Nature, 256:495(1975), or by recombinant DNA methods (U.S. Pat. No. 4,816,567).“Monoclonal antibodies” can also be isolated from phage antibodylibraries using the techniques described in Clackson et al., Nature352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597 (1991),for example.

The term anti-DEspR “antibody fragment” refers to a protein fragmentthat comprises at least an antigen binding site of the intact antibodyand thus retaining the ability to bind antigen. Examples of antibodyfragments encompassed by the term antibody fragment include: (i) the Fabfragment, having V_(L), C_(L), V_(H) and C_(H)1 domains; (ii) the Fab′fragment, which is a Fab fragment having one or more cysteine residuesat the C-terminus of the C_(H)1 domain; (iii) the Fd fragment havingV_(H) and C_(H)1 domains; (iv) the Fd′ fragment having V_(H) and C_(H)1domains and one or more cysteine residues at the C-terminus of the CH1domain; (v) the Fv fragment having the V_(L) and V_(H) domains of asingle arm of an antibody; (vi) the dAb fragment (Ward et al., Nature341, 544-546 (1989)) which consists of a V_(H) domain; (vii) isolatedCDR regions; (viii) F(ab')₂ fragments, a bivalent fragment including twoFab' fragments linked by a disulphide bridge at the hinge region; (ix)single chain antibody molecules (e.g., single chain Fv; scFv) (Bird etal., Science 242:423-426 (1988); and Huston et al., PNAS (USA)85:5879-5883 (1988)); (x) “diabodies” with two antigen binding sites,comprising a heavy chain variable domain (V_(H)) connected to a lightchain variable domain (V_(L)) in the same polypeptide chain (see, e.g.,EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad. Sci.USA, 90:6444-6448 (1993)); (xi) “linear antibodies” comprising a pair oftandem Fd segments (V_(H)-C_(H)1⁻V_(H) ⁻C_(H)1) which, together withcomplementary light chain polypeptides, form a pair of antigen bindingregions (Zapata et al. Protein Eng. 8(10):1057-1062 (1995); and U.S.Pat. No. 5,641,870).

In the hybridoma method of making an anti-DEspR monoclonal antibody, amouse or other appropriate host animal, such as a hamster or macaquemonkey, is immunized as described herein to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the DEspR protein or fragment thereof used for immunization.Alternatively, lymphocytes can be immunized in vitro. Lymphocytes thenare fused with myeloma cells using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Preferred myeloma cells are those that fuse efficiently, support stablehigh-level production of antibody by the selected antibody-producingcells, and are sensitive to a medium such as HAT medium. Among these,preferred myeloma cell lines are murine myeloma lines, such as thosederived from MOPC-21 and MPC-11 mouse tumors available from the SalkInstitute Cell Distribution Center, San Diego, Calif. USA, and SP-2 orX63-Ag8-653 cells available from the American Type Culture Collection,Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma celllines also have been described for the production of human monoclonalantibodies (Kozbor, J. Immunol , 133:3001 (1984); Brodeur et al.,Monoclonal Antibody Production Techniques and Applications, pp. 51-63(Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against the antigen.Preferably, the binding specificity of monoclonal antibodies produced byhybridoma cells is determined by immunoprecipitation or by an in vitrobinding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones can besubcloned by limiting dilution procedures and grown by standard methods(Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986)). Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, thehybridoma cells can be grown in vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of the monoclonal antibodies). The hybridomacells serve as a preferred source of such DNA. Once isolated, the DNAcan be placed into expression vectors, which are then transfected intohost cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. Recombinant production of antibodies isdescribed in more detail below.

Anti-DEspR Hybridomas and Monoclonal Antibodies Thereof

In certain aspects described herein, anti-DEspR monoclonal antibodiesinclude, but are not limited to, the monoclonal anti-DEspR antibody7C5B2 produced or expressed by the hybridoma 7C5B2 described herein, andreferred to as the “7C5B2 antibody,” and derivatives or antigen-bindingfragments thereof, including, for example, a “7C5B2 variable heavychain,” or a “7C5B2” variable light chain.

As described herein, the 7C5B2 hybridoma produces a monoclonal antibody,termed herein as the “7C5B2 anti-DEspR antibody” or “7C5B2 antibody,”that is highly specific for DEspR and can potently inhibit DEspRbiological activity. The biological characteristics of the 7C5B2anti-DEspR antibody render it particularly useful for the compositionsand methods described herein, including therapeutic and diagnosticapplications. Accordingly, sequence analysis of the 7C5B2 antibody wasperformed, as described herein, to identify the heavy and light chainvariable domain sequences, and complementarity determining region (CDR)sequences, of the 7C5B2 antibody for use in the compositions and methodsdescribed herein.

Throughout the present specification and claims, the numbering of theresidues in an immunoglobulin heavy chain is that of the EU index as inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991), which is also available on the world wide web, and is expresslyincorporated herein in its entirety by reference. The “EU index as inKabat” refers to the residue numbering of the human IgG 1 EU antibody.

As used herein, “antibody variable domain” refers to the portions of thelight and heavy chains of antibody molecules that include amino acidsequences of Complementarity Determining Regions (CDRs; i.e., CDR1,CDR2, and CDR3), and Framework Regions (FRs). V_(H) refers to thevariable domain of the heavy chain. V_(L) refers to the variable domainof the light chain. According to the methods used herein, the amino acidpositions assigned to CDRs and FRs can be defined according to Kabat(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991)). Amino acid numbering ofantibodies or antigen binding fragments is also according to that ofKabat.

As used herein, the term “Complementarity Determining Regions” (CDRs),i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of anantibody variable domain the presence of which are necessary for antigenbinding Each variable domain typically has three CDR regions identifiedas CDR1, CDR2 and CDR3. Each complementarity determining region cancomprise amino acid residues from a “complementarity determining region”as defined by Kabat (i.e., about residues 24-34 (L1), 50-56 (L2) and89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2)and 95-102 (H3) in the heavy chain variable domain; Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop” (i.e., about residues 26-32(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain and26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variabledomain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In someembodiments, a complementarity determining region can include aminoacids from both a CDR region defined according to Kabat and ahypervariable loop.

The nucleotide sequence encoding the V_(H) or variable domain of theheavy chain of the 7C5B2 antibody, as obtained by sequence analysis ofsequences obtained from the 7C5B2 hybridoma, is:CAGGTGCAACTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATTACCTGCACTGTCTCTGGGTTCTCATTAACCAGCTATGATATAAGCTGGATTCGCCAGCCACCAGGAAAGGGTCTGGAGTGGCTTGGAGTAATATGGACTGGTGGAGGCACAAATTATAATTCAGCTTTCATGTCCAGACTGAGCATCAGCAAGGACAACTCCAAGAGCCAAGTTTTCTTAAAAATGAACAGTCTGCAAACTGATGACACAGCCATATATTACTGTGTAAGAGATCGGGATTACGACGGGTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGT CACCGTCTCCTCA (SEQ ID NO:3).

The corresponding amino acid of the V_(H) domain of the 7C5B2 antibodyis: QVQL KESGPGLVAPSQSLSITCTVSGFSLTSYDISWIRQPPGKGLEWLGVIWTGGGTNYNSAFMSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCV RDRDYDGWYFDVWGAGTTVTVSS(SEQ ID NO: 4).

The 10 amino acid complementarity determining region 1 or CDR1 of theV_(H) domain of the 7C5B2 antibody is: GFSLTSYDIS (SEQ ID NO: 5). The 16amino acid CDR2 of the V_(H) domain of the 7C5B2 antibody is:VIWTGGGTNYNSAFMS (SEQ ID NO: 6). The 11 amino acid CDR2 of the V_(H)domain of the 7C5B2 antibody is: DRDYDGWYFDV (SEQ ID NO: 7).

The nucleotide sequence encoding the V_(L) or variable domain of thelight chain of the 7C5B2 antibody, as obtained by sequence analysis ofsequences obtained from the 7C5B2 hybridoma, is:GATGTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 8).

The corresponding amino acid of the V_(L) domain of the 7C5B2 antibodyis: DVLM TQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQG SHVPYTFGGGTKLEIK (SEQ IDNO: 9).

The 16 amino acid complementarity determining region 1 or CDR1 of theV_(L) domain of the 7C5B2 antibody is: RSSQSIVHSNGNTYLE (SEQ ID NO: 10).The 7 amino acid CDR2 of the V_(L) domain of the 7C5B2 antibody is:KVSNRFS (SEQ ID NO: 11). The 9 amino acid CDR2 of the V_(L) domain ofthe 7C5B2 antibody is: FQGSHVPYT (SEQ ID NO: 12).

As shown in Table 1, sequence analysis of the heavy and light chainvariable regions of the 7C5B2 antibody indicates strong homology tohuman germline sequences:

TABLE 1 Antibody Sequence Analysis^(a) H Chain L Chain CDR 1 Length 10aa16aa CDR 2 Length 16aa 7aa CDR 3 Length 11aa 9aa Closest HumanIGHV4-59*01 (64%) IGKV2-30*01 (82%) Germline^(b) Closest Human FW1^(b)IGHV4-31*01 (84%) IGKV2-30*01 (78%) Closest Human FW2^(b) IGHV4-61*01(93%) IGKV2-40*01 (93%) Closest Human FW3^(b) IGHV3-66*01 (60%)IGKV2-30*01 (97%) Closest Human J^(b) IGHJ6 (91%) IGKJ2 (90%) ^(a)CDRdefinitions and sequence numbering according to Kabat ^(b)Germline ID(s)indicated followed by % homology

Accordingly, in some embodiments of the aspects provided herein, theheavy and/or light chain variable domain(s) sequence(s) of the 7C5B2antibody, i.e., SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 8, and/or SEQ IDNO: 9 can be used to generate, for example, humanized antibodies, asdescribed elsewhere herein.

In some aspects, monoclonal antibodies that specifically bind to DEspRare provided having one or more biological characteristics of the 7C5B2monoclonal antibody. As used herein, an antibody having a “biologicalcharacteristic” of a designated antibody, such as the 7C5B2 antibody, isone that possesses one or more of the biological characteristics of thatantibody which distinguish it from other antibodies that bind to thesame antigen.

Accordingly, in some such embodiments of these aspects, having abiological characteristic of the 7C5B2 monoclonal antibody can includehaving an ED₅₀ value (i.e., the dose therapeutically effective in 50% ofthe population) at or around the ED₅₀ value of the 7C5B2 antibody forthe given population; having an EC₅₀ value (i.e., the dose that achievesa half-maximal inhibition of a given parameter or phenotype) at oraround the EC₅₀ value of the 7C5B2 antibody for a given parameter orphenotye. The effects of any particular dosage can be monitored by asuitable bioassay. For example, in some embodiments of these aspects,the given parameter or phenotype to be inhibited by the antibody thatspecifically binds to DEspR and has one or more biologicalcharacteristics of the 7C5B2 antibody can include, but is not limitedto, the mean total tube number in an in vitro tubulogenesis assay, themean total tube length in an in vitro tubulogenesis assay, the meannumber of branching points in an in vitro tubulogenesis assay, the meannumber of vessel connections in an in vitro tubulogenesis assay, and/ortumor cell invasiveness.

Accordingly, in those embodiments where the phenotype to be inhibited ismean total tube length, as measured using an in vitro tubulogenesisassay, the EC₅₀ value of the monoclonal antibody having a biologicalcharacteristic of the 7C5B2 monoclonal antibody is 10 nM or less, 9 nMor less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM orless, 3 nM or less, 2 nM or less, or 1 nM or less. In some suchembodiments, the EC₅₀ value of the monoclonal antibody is in the rangeof 3.0-5.0 nM, in the range of 3.1-4.9 nM, in the range of 3.2-4.8 nM,in the range of 3.3-4.7 nM, in the range of 3.4-4.6 nM, in the range of3.5-4.5 nM, in the range of 3.6-4.4 nM, in the range of 3.7-4.3 nM, inthe range of 3.8-4.2 nM, or in the range of 3.9-4.1 nM. In someembodiments, the EC₅₀ value for inhibiting mean total tube length of themonoclonal antibody having a biological characteristic of the 7C5B2monoclonal antibody is in the range of 3.8 nM-4.8 nM.

For example, in those embodiments where the phenotype to be inhibited isnumber of branch points, as measured using an in vitro tubulogenesisassay, the EC₅₀ value of the monoclonal antibody having a biologicalcharacteristic of the 7C5B2 monoclonal antibody is 10 nM or less, 9 nMor less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM orless, 3 nM or less, 2 nM or less, or 1 nM or less. In some suchembodiments, the EC₅₀ value of the monoclonal antibody is in the rangeof 3.0-5.0 nM, in the range of 3.1-4.9 nM, in the range of 3.2-4.8 nM,in the range of 3.3-4.7 nM, in the range of 3.4-4.6 nM, in the range of3.5-4.5 nM, in the range of 3.6-4.4 nM, in the range of 3.7-4.3 nM, inthe range of 3.8-4.2 nM, or in the range of 3.9-4.1 nM. In someembodiments, the EC₅₀ value for inhibiting total number of branch pointsof the monoclonal antibody having a biological characteristic of the7C5B2 monoclonal antibody is in the range of 3.4 nM-4.5 nM, in the rangeof 3.5 nM-4.4 nM, in the range of 3.6 nM-4.3 nM, in the range of 3.7nM-4.2 nM, in the range of 3.8 nM-4.1 nM, in the range of 3.9 nM-4.0 nM.

For example, in those embodiments where the phenotype to be inhibited istumor cell invasiveness, as measured in vitro, the EC₅₀ value of themonoclonal antibody having a biological characteristic of the 7C5B2monoclonal antibody is 10 nM or less, 9 nM or less, 8 nM or less, 7 nMor less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM orless, or 1 nM or less. In some such embodiments, the EC₅₀ value of themonoclonal antibody is in the range of 3.0-5.0 nM, in the range of3.1-4.9 nM, in the range of 3.2-4.8 nM, in the range of 3.3-4.7 nM, inthe range of 3.4-4.6 nM, in the range of 3.5-4.5 nM, in the range of3.6-4.4 nM, in the range of 3.7-4.3 nM, in the range of 3.8-4.2 nM, orin the range of 3.9-4.1 nM. In some embodiments, the EC₅₀ value forinhibiting tumor cell invasiveness of the monoclonal antibody having abiological characteristic of the 7C5B2 monoclonal antibody is in therange of 3.2 nM-3.9 nM, in the range of 3.3 nM-3.8 nM, 3.4 nM-3.7 nM, orin the range of 3.5 nM-3.6 nM.

In some embodiments of the aspects described herein, anti-DEspRantibodies for use in the compositions and methods described hereininclude monoclonal antibodies that bind to the same epitope or epitopesof DEspR as the monoclonal anti-DEspR 7C5B2 antibody.

In other aspects described herein, anti-DEspR antibodies for use in thecompositions and methods described herein include: the monoclonalanti-DEspR antibody 7C5C5 produced or expressed by the hybridoma 7C5C5described herein, referred to as the “7C5C5 antibody,” and derivativesor fragments thereof; monoclonal antibodies that bind to the sameepitope or epitopes of DEspR as the monoclonal anti-DEspR 7C5C5antibody; the monoclonal anti-DEspR antibody 5G12E8 produced orexpressed by the hybridoma5G12E8described herein, referred to as the“5G12E8 antibody,” and derivatives or fragments thereof; monoclonalantibodies that bind to the same epitope or epitopes of DEspR as themonoclonal anti-DEspR 5G12E8 antibody; and monoclonal antibodiesproduced by hybridomas 2E4A8, 2E4B11, 2E4H10, 8E7D11, 8E2F6, E2G4 and8E7F8.

In addition to generation and production via hybridomas, antibodies orantibody fragments that specifically bind DEspR can be isolated fromantibody phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature,352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)describe the isolation of murine and human antibodies, respectively,using phage libraries. Subsequent publications describe the productionof high affinity (nM range) human antibodies by chain shuffling (Markset al., Bio/Technology, 10:779-783 (1992)), as well as combinatorialinfection and in vivo recombination as a strategy for constructing verylarge phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266(1993)). Thus, these techniques are viable alternatives to traditionalmonoclonal antibody hybridoma techniques for isolation of monoclonalantibodies.

The DNA sequences encoding the antibodies or antibody fragment thatspecifically bind DEspR also can be modified, for example, bysubstituting the coding sequence for human heavy- and light-chainconstant domains in place of the homologous murine sequences (U.S. Pat.No. 4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851(1984)), or by covalently joining to the immunoglobulin coding sequenceall or part of the coding sequence for a non-immunoglobulin polypeptide,as also described elsewhere herein.

Such non-immunoglobulin polypeptides can be substituted for the constantdomains of an antibody, or they can be substituted for the variabledomains of one antigen-combining site of an antibody to create achimeric bivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

Humanized and Human Antibodies

Provided herein, in some aspects, are humanized anti-DEspR antibodiesfor use in the compositions and methods described herein. Humanizedforms of non-human (e g., murine) antibodies refer to chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, Fv framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies can compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, a humanized antibody can comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also can comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522-525 (1986); Riechmannet al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

A humanized antibody has one or more amino acid residues introduced intoit from a source which is non-human. These non-human amino acid residuesare often referred to as “import” residues, which are typically takenfrom an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.,Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567) where substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies. In some embodiments, humanized antibodiescomprising one or more variable domains comprising the amino acidsequence of the variable heavy (SEQ ID NO: 4) and/or variable light (SEQID NO: 9) chain domains of the murine anti-DEspR antibody 7C5B2, areprovided.

Accordingly, in some embodiments of the aspects described herein, one ormore heavy and/or one or more light chain CDR regions of a humanizedanti-DEspR antibody or antibody fragment thereof comprises a sequence ofthe 7C5B2 antibody described herein. In some such embodiments, the oneor more variable heavy chain CDR regions comprises a sequence selectedfrom the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:7. In some such embodiments, the one or more variable light chain CDRregions comprises a sequence selected from the group consisting of SEQID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some such embodiments,the one or more variable heavy chain CDR regions comprises a sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQID NO: 7, and the one or more variable light chain CDR regions comprisesa sequence selected from the group consisting of SEQ ID NO: 10, SEQ IDNO: 11, or SEQ ID NO: 12.

In some embodiments of the aspects described herein, a humanizedanti-DEspR monoclonal antibody comprises mutated human IgG 1 frameworkregions and one or more heavy and/or one or more light chain CDR regionsfrom the murine anti-human DEspR monoclonal antibody 7C5B2, describedherein, that blocks binding of human DEspR to its ligands. In some suchembodiments, the one or more variable heavy chain CDR regions comprisesa sequence selected from the group consisting of SEQ ID NO: 5, SEQ IDNO: 6, or SEQ ID NO: 7. In some such embodiments, the one or morevariable light chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. Insome such embodiments, the one or more variable heavy chain CDR regionscomprises a sequence selected from the group consisting of SEQ ID NO: 5,SEQ ID NO: 6, or SEQ ID NO: 7, and the one or more variable light chainCDR regions comprises a sequence selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

In some embodiments, a humanized anti-DEspR monoclonal antibodycomprises mutated human IgG4 framework regions and one or more heavyand/or one or more light chain CDR regions from the murine anti-humanDEspR monoclonal antibody 7C5B2, described herein, that blocks bindingof human DEspR to its ligands. In some such embodiments, the one or morevariable heavy chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In somesuch embodiments, the one or more variable light chain CDR regionscomprises a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID NO: 12. In some such embodiments, the oneor more variable heavy chain CDR regions comprises a sequence selectedfrom the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:7, and the one or more variable light chain CDR regions comprises asequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO:11, or SEQ ID NO: 12.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the aminoacid sequences of the variable heavy and light chain domains of a rodentantibody, such as that of the 7C5B2 antibody (SEQ ID NO: 4 and SEQ IDNO: 9, respectively), are screened against the entire library of knownhuman variable-domain sequences. The human sequence which is closest tothat of the rodent is then accepted as the human framework (FR) for thehumanized antibody (Sims et al., J. Immunol , 151:2296 (1993); Chothiaet al., J. Mol. Biol., 196:901 (1987)). Another method uses a particularframework derived from the consensus sequence of all human antibodies ofa particular subgroup of light or heavy chains. The same framework canbe used for several different humanized antibodies (Carter et al., Proc.Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol ,151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties,for example, the anti-angiogenic properties of the anti-DEspR antibody7C5B2 described herein. To achieve this goal, according to a preferredmethod, humanized antibodies are prepared by a process of analysis ofthe parental sequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the CDR residues aredirectly and most substantially involved in influencing antigen binding.

Exemplary humanized antibodies and affinity matured variants thereofdirected against the VEGF antigen are described in, for example, U.S.Pat. No. 6,884,879 issued Feb. 26, 2005.

Alternatively, it is now possible to produce transgenic animals (e.g.,mice) that are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. For example, it has been described that thehomozygous deletion of the antibody heavy-chain joining region (J_(H))gene in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of the humangerm-line immunoglobulin gene array in such germ-line mutant mice willresult in the production of human antibodies upon antigen challenge.See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551(1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann etal., Year in Immuno , 7:33 (1993); and Duchosal et al. Nature 355:258(1992).

Alternatively, phage display technology (McCafferty et al., Nature348:552-553 (1990)) can be used to produce human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell. Phage display can be performed in avariety of formats; for their review see, e.g., Johnson, Kevin S, andChiswell, David J., Current Opinion in Structural Biology 3:564-571(1993). Several sources of V-gene segments can be used for phagedisplay. Clackson et al., Nature, 352:624-628 (1991) isolated a diversearray of anti-oxazolone antibodies from a small random combinatoriallibrary of V genes derived from the spleens of immunized mice. Arepertoire of V genes from unimmunized human donors can be constructedand antibodies to a diverse array of antigens (including self-antigens)can be isolated essentially following the techniques described by Markset al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J.12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and 5,573,905.

Human antibodies can also be generated by in vitro activated B cells(see U.S. Pat. Nos. 5,567,610 and 5,229,275).

Design & Generation of Composite Human Antibodies

In some embodiments of the aspects described herein, composite humanantibody technology that generates de-immunized 100% engineered humanantibodies at the outset can be used to prepare humanized compositeanti-DEspR antibodies for use in the compositions and methods describedherein, using, for example, a technology as described by Antitope.

Briefly, as used herein, “composite human antibodies” comprise multiplesequence segments (“composites”) derived from V-regions of unrelatedhuman antibodies that are selected to maintain monoclonal antibodysequences critical for antigen binding of the starting murine precursoranti-human DEspR monoclonal antibody, such as 7C5B2 antibody, and whichhave all been filtered for the presence of potential T-cell epitopesusing “in silico tools” (Holgate & Baker, 2009). The close fit of humansequence segments with all sections of the starting antibody V regionsand the elimination of CD4+ T cell epitopes from the outset allow thistechnology to circumvent immunogenicity in the development of ‘100%engineered human’ therapeutic antibodies while maintaining optimalaffinity and specificity through the prior analysis of sequencesnecessary for antigen-specificity (Holgate & Baker 2009).

As described herein, structural models of mouse anti-hDEspR antibody Vregions were produced using Swiss PDB and analysed in order to identifyimportant “constraining” amino acids in the V regions that were likelyto be essential for the binding properties of the antibody. Residuescontained within the CDRs (using Kabat definition) together with anumber of framework residues were considered to be important. Both theV_(H) and V_(L) (V_(K)) sequences of anti-hDEspR, as described herein asSEQ ID NO: 4 and SEQ ID NO: 9, comprise typical framework residues andthe CDR1, CDR2, and CDR3 motifs are comparable to many murineantibodies, as described elsewhere herein.

From the above analysis, it was determined that composite humansequences of anti-hDEspR can be created with a wide latitude ofalternatives outside of CDRs but with only a narrow menu of possiblealternative residues within the CDR sequences. Analysis indicated thatcorresponding sequence segments from several human antibodies could becombined to create CDRs similar or identical to those in the murinesequences. For regions outside of and flanking the CDRs, a wideselection of human sequence segments were identified as possiblecomponents of novel anti-DEspR composite human antibody V regions foruse with the compositions and methods described herein (see, forexample, Table 1).

Based upon these analyses, a large preliminary set of sequence segmentsthat could be used to create novel anti-DEspR composite human antibodyvariants were selected and analysed using iTope™ technology for insilico analysis of peptide binding to human MHC class II alleles (Perryet al 2008), and using the TCED™ (T Cell Epitope Database) of knownantibody sequence-related T cell epitopes (Bryson et al 2010). Sequencesegments that were identified as significant non-human germline bindersto human MHC class II or that scored significant hits against the TCED™were discarded. This resulted in a reduced set of segments, andcombinations of these were again analysed, as above, to ensure that thejunctions between segments did not contain potential T cell epitopes.Selected segments were then combined to produce heavy and light chain Vregion sequences for synthesis.

Accordingly, provided herein are variable heavy and light chainsequences for use in anti-DEspR composite human antibody or engineeredhuman antibody production. In some embodiments, an anti-DEspR compositehuman antibody can comprise a variable heavy (V_(H)) chain amino acidsequence selected from the group consisting of:QVQLQESGPGLVKPSQTLSLTCTVSGFSLTSYDISWIRQPPGKGLEWLGVIWTGGGTNYNSAFMSRLTISKDNSKSTVYLQMNSLRAEDTAIYYCVRDRDYDGWYFDVWGQGTTVTVSS (SEQ ID NO: 13);

-   QVQLQESGPGLVKPSQTLSLTCTVSGFSLTSYDISWIRQPPGKGLEWL    GVIWTGGGTNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAIYY    CVRDRDYDGWYFDVWGQGTTVTVSS (SEQ ID NO: 14);-   QVQLQESGPGLVKPSQTLSLTCTVSGFSLTSYDISWIRQPPGKGLEW    LGVIWTGGGTNYNSAFMSRFTISKDNSKNTVYLQMNSLRAEDTAIY    YCVRDRDYDGWYFDVWGQGTTVTVSS (SEQ ID NO: 15);-   QVQLQESGPGLVKPSQTLSLTCTVSGFSLTSYDISWIRQPPGKGLEWL    GVIWTGGGTNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAVYY    CVRDRDYDGWYFDVWGQGTTVTVSS (SEQ ID NO:16); and-   QVQLQESGPGLVKPSQTLSLTCTVSGFSLTSYDISWIRQPPGKGLEWL    GVIWTGGGTNYNSAFMSRFTISKDNSKNTVYLQMNSLRAEDTAVYY    CVRDRDDYDGWYFDVWGQGTTVTVSS (SEQ ID NO: 17).

In some embodiments, an anti-DEspR composite human antibody can comprisea variable light (V_(L)) chain amino acid sequence selected from thegroup consisting of: DVLMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSH VPYTFGQGTKLEIK (SEQ ID NO:18) and DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWYLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYY CFQGSHVPYTFGQGTKLEIK (SEQID NO: 19).

In some embodiments, an anti-DEspR composite human antibody can comprisea heavy chain CDR1 region comprising an amino acid sequence of SEQ IDNO: 5. In some embodiments, an anti-DEspR composite human antibody cancomprise a heavy chain CDR2 region comprising an amino acid sequence ofSEQ ID NO: 6. In some embodiments, an anti-DEspR composite humanantibody can comprise a heavy chain CDR3 region comprising an amino acidsequence of SEQ ID NO: 7.

In some embodiments, an anti-DEspR composite human antibody can comprisea light chain CDR1 region comprising a sequence of SEQ ID NO: 10. Insome embodiments, an anti-DEspR composite human antibody can comprise alight chain CDR2 region comprising an amino acid sequence of SEQ ID NO:11. In some embodiments, an anti-DEspR composite human antibody cancomprise a light chain CDR3 region comprising an amino acid sequence ofSEQ ID NO: 12.

Antibody Fragments

In some embodiments of the aspects described herein, an antibodyspecific for DEspR, such as, for example the anti-DEspR 7C5B2 antibody;an anti-DEspR antibody comprising one or more heavy chain CDR regionscomprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO: 6, or SEQ ID NO: 7; an anti-DEspR antibody comprising oneor more light chain CDR regions comprises a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; ananti-DEspR composite human antibody comprising a variable heavy (V_(H))chain amino acid sequence selected from the group consisting of SEQ IDNO: 4 and SEQ ID NO: 13-SEQ ID NO: 17; or an anti-DEspR composite humanantibody comprising a variable light (V_(L)) chain amino acid sequenceselected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, andSEQ ID NO: 19 can be treated or processed into an antibody fragmentthereof.

Various techniques have been developed and are available for theproduction of antibody fragments. Traditionally, these fragments werederived via proteolytic digestion of intact antibodies (see, e.g.,Morimoto et al., Journal of Biochemical and Biophysical Methods24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However,these fragments can now be produced directly by recombinant host cells.For example, antibody fragments can be isolated from the antibody phagelibraries discussed above. Alternatively, Fab'-SH fragments can bedirectly recovered from E. coli and chemically coupled to form F(ab')₂fragments (Carter et al., Bio/Technology 10:163-167 (1992)). Accordingto another approach, F(ab')₂fragments can be isolated directly fromrecombinant host cell culture. Other techniques for the production ofantibody fragments will be apparent to the skilled practitioner. Inother embodiments, the antibody fragment of choice is a single chain Fvfragment (scFv). See WO 93/16185.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fab fragment comprising V_(L),C_(L), V_(H) and C_(H)1 domains. Fab fragments comprise a variable andconstant domain of the light chain and a variable domain and the firstconstant domain (C_(H)1) of the heavy chain. In some such embodiments,the V_(H) domain is selected from the group consisting of SEQ ID NO: 4and SEQ ID NO: 13-SEQ ID NO: 17. In some such embodiments, the V_(H)domain comprises one or more heavy chain CDR regions comprising asequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO:6, and SEQ ID NO: 7. In some such embodiments, the V_(L) domain isselected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, andSEQ ID NO: 19. In some such embodiments, the V_(L) domain comprises oneor more light chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fab' fragment, which is a Fabfragment having one or more cysteine residues at the C-terminus of theC_(H)1 domain.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fd fragment comprising V_(H) andC_(H)1 domains. In some such embodiments, the V_(H) domain is selectedfrom the group consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID NO:17. In some such embodiments, the V_(H) domain comprises one or moreheavy chain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a Fd' fragment comprising V_(H) andC_(H)1 domains and one or more cysteine residues at the C-terminus ofthe C_(H)1 domain. In some such embodiments, the V_(H) domain isselected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQID NO: 17. In some such embodiments, the V_(H) domain comprises one ormore heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

Single-chain Fv or scFv antibody fragments comprise the V_(H) and V_(L)domains of antibody, such that these domains are present in a singlepolypeptide chain. Generally, a Fv polypeptide further comprises apolypeptide linker between the V_(H) and V_(L) domains, which enablesthe scFv to form the desired structure for antigen binding. For a reviewof scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315(1994). Accordingly, in some embodiments of the aspects describedherein, a human DEspR-specific antibody fragment is a Fv fragmentcomprising the V_(L) and V_(H) domains of a single arm of an antibody.In some such embodiments, the V_(H) domain is selected from the groupconsisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID NO: 17. In some suchembodiments, the V_(H) domain comprises one or more heavy chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some such embodiments, theV_(L) domain is selected from the group consisting of SEQ ID NO: 9, SEQID NO: 18, and SEQ ID NO: 19. In some such embodiments, the V_(L) domaincomprises one or more light chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, orSEQ ID NO: 12.

The term diabodies refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H) and V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

Accordingly, in some embodiments of the aspects described herein, ahuman DEspR-specific antibody fragment is a diabody comprising twoantigen binding sites, comprising a heavy chain variable domain (V_(H))connected to a light chain variable domain (V_(L)) in the samepolypeptide chain. In some such embodiments, the V_(H) domain isselected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQID NO: 17. In some such embodiments, the V_(H) domain comprises one ormore heavy chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. Insome such embodiments, the V_(L) domain is selected from the groupconsisting of SEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO: 19. In somesuch embodiments, the V_(L) domain comprises one or more light chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a dAb fragment comprising a V_(H)domain. In some such embodiments, the V_(H) domain is selected from thegroup consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID NO: 17. Insome such embodiments, the V_(H) domain comprises one or more heavychain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment comprises isolated CDR regions. In somesuch embodiments, the isolated CDR region comprises one or more heavychain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some suchembodiments, the isolated CDR region comprises one or more light chainCDR regions comprising a sequence selected from the group consisting ofSEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

In some embodiments of the aspects described herein, the humanDEspR-specific antibody fragment is a F(ab')₂ fragment, which comprisesa bivalent fragment comprising two Fab' fragments linked by a disulphidebridge at the hinge region.

Linear antibodies refers to the antibodies as described in Zapata etal., Protein Eng., 8(10):1057-1062 (1995). Briefly, these antibodiescomprise a pair of tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

In some embodiments of the aspects described herein, a humanDEspR-specific antibody fragment is a linear antibody comprising a pairof tandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1) which, together withcomplementary light chain polypeptides, form a pair of antigen bindingregions. In some such embodiments, the V_(H) domain is selected from thegroup consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID NO: 17. Insome such embodiments, the V_(H) domain comprises one or more heavychain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some suchembodiments, the V_(L) domain is selected from the group consisting ofSEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO: 19. In some suchembodiments, the V_(L) domain comprises one or more light chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

In other embodiments of these aspects, a human DEspR-specific antibodyfragment has specificity for the same epitope as the monoclonalanti-DEspR antibody 7C5B2, described herein, and produced by hybridoma7C5B2.

Some further examples of DEspR-inhibiting antibodies are described inPCT/US2005/041594, the contents of which are incorporated herein byreference in their entirety.

Other Amino Acid Sequence Modifications

In some embodiments of the aspects described herein, amino acid sequencemodification(s) of the antibodies or antibody fragments thereof specificfor DEspR described herein are contemplated. For example, it can bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics, e.g., binding specificity,inhibition of biological activity. The amino acid changes also can alterpost-translational processes of the antibody, such as changing thenumber or position of glycosylation sites.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells Science,244:1081-1085 (1989). Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed antibodyvariants are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includeantibody with an N-terminal methionyl residue or the antibody fused to acytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated for use in the antibodies or antibodyfragments thereof specific for DEspR described herein.

Substantial modifications in the biological properties of the antibodiesor antibody fragments thereof specific for DEspR are accomplished byselecting substitutions that differ significantly in their effect onmaintaining (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a sheet or helical conformation, (b)the charge or hydrophobicity of the molecule at the target site, or (c)the bulk of the side chain. Amino acids can be grouped according tosimilarities in the properties of their side chains (in A. L. Lehninger,in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York(1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe(F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser (S), Thr (T),Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp (D), Glu (E); (4)basic: Lys (K), Arg (R), His (H).

Alternatively, naturally occurring residues can be divided into groupsbased on common side-chain properties: (1) hydrophobic: Norleucine, Met,Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues thatinfluence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

Any cysteine residue not involved in maintaining the proper conformationof the antibodies or antibody fragments thereof specific for DEspR alsocan be substituted, generally with serine, to improve the oxidativestability of the molecule and prevent aberrant crosslinking Conversely,cysteine bond(s) can be added to the antibody to improve its stability(particularly where the antibody is an antibody fragment such as an Fvfragment).

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a parentantibody (e.g., the monoclonal anti-DEspR antibody 7C5B2, or a humanizedor human antibody or antibody fragment thereof specific for DEspR, asprovided herein). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.,6-7 sites) are mutated to generate all possible amino substitutions ateach site. The antibody variants thus generated are displayed in amonovalent fashion from filamentous phage particles as fusions to thegene III product of M13 packaged within each particle. Thephage-displayed variants are then screened for their biological activity(e.g. binding affinity) as herein disclosed. In order to identifycandidate hypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding.

Alternatively, or additionally, it can be beneficial to analyze acrystal structure of the antigen-antibody complex to identify contactpoints between the antibody or antibody fragments thereof specific forDEspR and human DEspR. Such contact residues and neighboring residuesare candidates for substitution according to the techniques elaboratedherein. Once such variants are generated, the panel of variants issubjected to screening as described herein and antibodies or antibodyfragments thereof with superior properties in one or more relevantassays can be selected for further development.

Another type of amino acid variant of the antibody alters the originalglycosylation pattern of the antibody. By altering is meant deleting oneor more carbohydrate moieties found in the antibody, and/or adding oneor more glycosylation sites that are not present in the antibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine can also be used.

Addition of glycosylation sites to the antibodies or antibody fragmentsthereof specific for DEspR is accomplished by altering the amino acidsequence such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationcan also be made by the addition of, or substitution by, one or moreserine or threonine residues to the sequence of the original antibody(for O-linked glycosylation sites).

Where the antibody comprises an Fc region, the carbohydrate attachedthereto can be altered. For example, antibodies with a maturecarbohydrate structure that lacks fucose attached to an Fc region of theantibody are described in US Pat Appl No US 2003/0157108 Al, Presta, L.See also US 2004/0093621 Al (Kyowa Hakko Kogyo Co., Ltd). Antibodieswith a bisecting N-acetylglucosamine (G1cNAc) in the carbohydrateattached to an Fc region of the antibody are referenced in WO03/011878,Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodieswith at least one galactose residue in the oligosaccharide attached toan Fc region of the antibody are reported in WO97/30087, Patel et al.See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.) concerningantibodies with altered carbohydrate attached to the Fc region thereof.

In some embodiments, it can be desirable to modify the antibodies orantibody fragments thereof specific for DEspR described herein withrespect to effector function, e.g., so as to enhance antigen-dependentcell-mediated cyotoxicity (ADCC) and/or complement dependentcytotoxicity (CDC) of the antibody. This can be achieved by introducingone or more amino acid substitutions in an Fc region of the antibody orantibody fragment thereof . Alternatively or additionally, cysteineresidue(s) can be introduced in the Fc region, thereby allowinginterchain disulfide bond formation in this region. The homodimericantibody thus generated can have improved internalization capabilityand/or increased complement-mediated cell killing and antibody-dependentcellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.176:1191-1195 (1992) and Shopes, B. J. Immunol 148:2918-2922 (1992).Homodimeric antibodies with enhanced anti-tumor activity can also beprepared using heterobifunctional cross-linkers as described in Wolff etal. Cancer Research 53:2560-2565 (1993). Alternatively, an antibody canbe engineered which has dual Fc regions and can thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al. Anti-CancerDrug Design 3:219-230 (1989).

For example, WO00/42072 (Presta, L.) describes antibodies with improvedADCC function in the presence of human effector cells, where theantibodies comprise amino acid substitutions in the Fc region thereof.Preferably, the antibody with improved ADCC comprises substitutions atpositions 298, 333, and/or 334 of the Fc region (Eu numbering ofresidues). Preferably the altered Fc region is a human IgG1 Fc regioncomprising or consisting of substitutions at one, two or three of thesepositions. Such substitutions are optionally combined withsubstitution(s) which increase Clq binding and/or CDC.

Antibodies with altered C1q binding and/or complement dependentcytotoxicity (CDC) are described in WO99/51642, U.S. Pat. Nos.6,194,551B1, 6,242,195B1, 6,528,624B1 and 6,538,124 (Idusogie et al.).The antibodies comprise an amino acid substitution at one or more ofamino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334 of theFc region thereof (Eu numbering of residues).

To increase the serum half life of the antibody specific for DEspRdescribed herein, one can incorporate a salvage receptor binding epitopeinto the antibody (especially an antibody fragment) as described in U.S.Pat. No. 5,739,277, for example. As used herein, the term “salvagereceptor binding epitope” refers to an epitope of the Fc region of anIgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) thatis responsible for increasing the in vivo serum half-life of the IgGmolecule.

Antibodies with improved binding to the neonatal Fc receptor (FcRn), andincreased half-lives, are described in WO00/42072 (Presta, L.) andUS2005/0014934A1 (Hinton et al.). These antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. For example, the Fc region can have substitutions at oneor more of positions 238, 250, 256, 265, 272, 286, 303, 305, 307, 311,312, 314, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, 428 or434 (Eu numbering of residues). The preferred Fc region-comprisingantibody variant with improved FcRn binding comprises amino acidsubstitutions at one, two or three of positions 307, 380 and 434 of theFc region thereof (Eu numbering of residues). In one embodiment, theantibody has 307/434 mutations.

Engineered antibodies specific for DEspR with three or more (preferablyfour) functional antigen binding sites are also contemplated (US ApplnNo. US2002/0004587 A1, Miller et al.).

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

Immunoconjugates

In some embodiments of these aspects, immunoconjugates comprising theantibody and antibody fragments specific for DEspR described herein areconjugated to an agent such as a chemotherapeutic agent, toxin (e.g. anenzymatically active toxin of bacterial, fungal, plant or animal origin,or fragments thereof), a small molecule, an siRNA, a nanoparticle, atargeting agent (e.g., a microbubble), or a radioactive isotope (i.e., aradioconjugate) can be used. Such immunoconjugates can be used, forexample, in diagnostic, theranostic, or targeting methods.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates are described herein. Enzymatically active toxins andfragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugate antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y and¹⁸⁶Re.

Conjugates of the antibodies specific for DEspR described herein and acytotoxic agent can be made using any of a variety of bifunctionalprotein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives ofimidoesters (such as dimethyl adipimidate HCL), active esters (such asdisuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azidocompounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazoniumderivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),diisocyanates (such as tolyene 2,6-diisocyanate), and bis-activefluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Forexample, a ricin immunotoxin can be prepared as described in Vitetta etal. Science 238: 1098 (1987). Carbon-14-labeled1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid(MX-DTPA) is an exemplary chelating agent for conjugation ofradionucleotide to the antibody. See WO94/11026.

In other embodiments, the DEspR-specific antibody or antibody fragmentthereof can be conjugated to a “receptor” (such as, for example,streptavidin) for utilization in tumor pretargeting wherein theantibody-receptor conjugate is administered to the subject, followed byremoval of unbound conjugate from the circulation using a clearing agentand then administration of a “ligand” (e.g. avidin) which is conjugatedto a cytotoxic agent (e.g. a radionucleotide). In some embodiments, theDEspR-specific antibody or antibody fragment thereof can be conjugatedto biotin, and the biotin conjugated antibody or antibody fragmentthereof can be further conjugated or linked to a streptavidin-bound or-coated agent, such as a streptavidin-coated microbubble, for use in,for example, molecular imaging of angiogenesis.

Immunoliposomes

The antibodies and antibody fragments thereof specific for DEspRdescribed herein can also be formulated as immunoliposomes. Liposomescontaining the antibody are prepared by methods known in the art, suchas described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688(1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); andU.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhancedcirculation time are disclosed in U.S. Pat. No. 5,013,556.

Particularly useful liposomes can be generated, for example, by thereverse phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab' fragments of the antibody of the invention can beconjugated to the liposomes as described in Martin et al. J. Biol. Chem.257: 286-288 (1982) via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome. SeeGabizon et a/. J. National Cancer Inst. 81(19)1484 (1989)

The hybridoma cell lines 7C5B2, 7C5C5, and 5G12E8 are being maintainedand stored.

Compositions and Therapeutic & Diganotic Uses of Anti-DEspR Antibodiesand Fragments Thereof

Certain aspects described herein are based, in part, on the discovery bythe inventors that DEspR plays contributes to adult tissue vascularity,as well as playing a critical role in angiogenesis during embryonicdevelopment, and further that DEspR is surpisingly expressed in certaintumor cells, cancer stem cells or stem-like cells, or tumor initiatingcells, as well as in tumor-surrounding blood vessels' endothelial cells,pericytes, and smooth muscle cells. The inventors further discoveredthat inhibition of DEspR, using DEspR-specific inhibitors, such as theanti-DEspR antibodies and antibody fragments thereof described herein,can inhibit a variety of parameters that characterize tumor metastasis,including cell invasiveness, tumor growth, such as tumor volume or tumormass, as well as parameters that characterize angiogenesis, includingneovessel tube length, neovessel branching, and formation of vesselinterconnections. The anti-DEspR antibodies and antibody fragmentsthereof described herein are further highly suitable for antibody-targetsonoporation and demonstrate enhanced penetration and efficacy whenadministered using, for example, ultrasound methods. In addition, theinventors have determined that DEspR serves as a diagnostic marker for avariety of disease conditions.

Anti-Angiogenic Therapeutics and Treatments

Angiogenesis is a process of tissue vascularization that involves boththe growth of new developing blood vessels into a tissue(neo-vascularization) and co-opting of existing blood vessels to atarget site. Blood vessels are the means by which oxygen and nutrientsare supplied to living tissues and waste products are removed fromliving tissue. Angiogenesis can be a critical biological process. Forexample, angiogenesis is essential in reproduction, development andwound repair. Conversely, inappropriate angiogenesis can have severenegative consequences. For example, it is only after solid tumors arevascularized as a result of angiogenesis that the tumors have asufficient supply of oxygen and nutrients that permit it to grow rapidlyand metastasize.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease, inhibition of angiogenesis,using the compositions and methods described herein, can reduce thedeleterious effects of the disease. Non-limiting examples includetumors, carotid artery disease, rheumatoid arthritis, diabeticretinopathy, inflammatory diseases, restenosis, and the like. Where thegrowth of new blood vessels is required to support growth of adeleterious tissue, inhibition of angiogenesis, using the compositionsand methods described herein, can reduce the blood supply to the tissueand thereby contribute to reduction in tissue mass based on blood supplyrequirements. Non-limiting examples include growth of tumors whereneovascularization is a continual requirement in order that the tumorgrowth beyond a few millimeters in thickness, and for the establishmentof solid tumor metastases. Another example is coronary plaqueenlargement.

There are a variety of diseases or disorders in which angiogenesis isbelieved to lead to negative consequences, referred to as pathologicalangiogenesis, or diseases or disorders dependent or modulated byangiogenesis, including but not limited to, inflammatory disorders suchas immune and non-immune inflammation, chronic articular rheumatism andpsoriasis, disorders associated with inappropriate or inopportuneinvasion of vessels such as diabetic retinopathy, neovascular glaucoma,restenosis, capillary proliferation in atherosclerotic plaques andosteoporosis, and cancer associated disorders, such as solid tumors,solid tumor metastases, angiofibromas, retrolental fibroplasia,hemangiomas, Kaposi sarcoma and the like cancers which requireneovascularization to support tumor growth. In a preferred embodiment ofthe aspects described herein, the methods are directed to inhibitingangiogenesis in a subject with cancer.

The antibodies and antibody fragments specific for DEspR describedherein, such as, for example the anti-DEspR 7C5B2 antibody; ananti-DEspR antibody comprising one or more heavy chain CDR regionscomprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO: 6, or SEQ ID NO: 7; an anti-DEspR antibody comprising oneor more light chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; ananti-DEspR composite human antibody comprising a variable heavy (V_(H))chain amino acid sequence selected from the group consisting of SEQ IDNO: 4 and SEQ ID NO: 13-SEQ ID NO: 17; or an anti-DEspR composite humanantibody comprising a variable light (V_(L)) chain amino acid sequenceselected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, andSEQ ID NO: 19, and fragments thereof, can be used in compositions andmethods of antiangiogenic therapy. These antiangiogenic therapies can beused as novel cancer treatment strategies aimed at inhibiting existingtumor blood vessels and development of tumor blood vessels required forproviding nutrients to support tumor growth. Because angiogenesis isinvolved in both primary tumor growth and metastasis, the antiangiogenictreatments using the antibodies and antibody fragments specific forDEspR described herein are capable of inhibiting the neoplastic growthof tumor at the primary site, as well as preventing micro- andmacro-metastasis of tumors at the secondary sites, therefore allowingattack of the tumors by other therapeutics.

Addtionally, the antibodies and antibody-fragments specific for DEspRdescribed herein, such as, for example the anti-DEspR 7C5B2 antibody; ananti-DEspR antibody comprising one or more heavy chain CDR regionscomprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO: 6, or SEQ ID NO: 7; an anti-DEspR antibody comprising oneor more light chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; ananti-DEspR composite human antibody comprising a variable heavy (V_(H))chain amino acid sequence selected from the group consisting of SEQ IDNO: 4 and SEQ ID NO: 13-SEQ ID NO: 17; or an anti-DEspR composite humanantibody comprising a variable light (V_(L)) chain amino acid sequenceselected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, andSEQ ID NO: 19, and fragments thereof, can be used in methods ofantimetastasis therapy. Such antimetastasis therapies provide novelcancer treatment strategies aimed at inhibiting concurrent inhibition oftumor vascularization and tumor cell invasiveness for treatment and/orinhibition of micrometastasis and macrometastasis, as further describedherein. Furthermore, since DEspR is also expressed in tumor cells,including cancer stem cells, as demonstrated herein, immunoconjugates ofDEspR specific antibodies or antibody fragments thereof, as describedherein, can be generated by conjugation to any agent such as a toxin,cytotoxic or pro-apoptotic agent, and can further inhibit tumor growthby directly targeting/killing tumor cells and cancer stem cells.

Accordingly, angiogenesis-dependent diseases and disorders that can betreated using the methods and compositions comprising antibodies andantibody fragments specific for DEspR described herein, such as, forexample, the anti-DEspR 7C5B2 antibody; an anti-DEspR antibodycomprising one or more heavy chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQID NO: 7; an anti-DEspR antibody comprising one or more light chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; an anti-DEspR compositehuman antibody comprising a variable heavy (V_(H)) chain amino acidsequence selected from the group consisting of SEQ ID NO: 4 and SEQ IDNO: 13-SEQ ID NO: 17; or an anti-DEspR composite human antibodycomprising a variable light (V_(L)) chain amino acid sequence selectedfrom the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO:19, and fragments thereof, are those diseases and disorders affected byvascular growth. In other words, an “angiogenesis-dependent disease ordisorder” refers to those diseases or disorders that are dependent on arich blood supply and blood vessel proliferation for the diseases'pathological progression (e.g., metastatic tumors), or diseases ordisorders that are the direct result of aberrant blood vesselproliferation (e.g., diabetic retinopathy and hemangiomas).

Non-limiting examples of angiogenesis-dependent diseases or disorderthat can be treated using the compositions and methods described hereininclude abnormal vascular proliferation, ascites formation, psoriasis,age-related macular degeneration, thyroid hyperplasia, preeclampsia,rheumatoid arthritis and osteoarthritis, carotid artery disease, vasovasorum neovascularization, vulnerable plaque neovascularization,neurodegenerative disorders, Alzheimer's disease, obesity, pleuraleffusion, atherosclerosis, endometriosis, diabetic/other retinopathies,ocular neovascularizations such as neovascular glaucoma and cornealneovascularization, disorders associated with inappropriate orinopportune invasion of vessels such as diabetic retinopathy, maculardegeneration, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi sarcoma, cancers whichrequire neovascularization to support tumor growth, etc.

Accordingly, described herein are methods of inhibiting angiogenesis ina tissue of a subject or individual having a disease or disorderdependent or modulated by angiogenesis, where the disease or disordercan be treated by the inhibition of angiogenesis. Generally, the methodscomprise administering to the subject a therapeutically effective amountof a composition comprising an angiogenesis-inhibiting amount of a DEspRinhibitor. In some embodiments, the methods further comprises selectingor diagnosing a subject having or at risk for a disease or disordermodulated by angiogenesis.

In some embodiments of these methods and all such methods describedherein, the DEspR inhibitor is an antibody or antibody fragment thereof.Accordingly, in some aspects, an anti-DEspR antibody orantibody-fragment thereof that is specific for a DEspR target isprovided, where the anti-DEspR antibody or antibody-fragment thereofspecifically binds to the DEspR target and reduces or inhibits DEspRbiological activity, thus inhibiting angiogenesis in the subject havinga disease or disorder dependent on angiogenesis.

In some such embodiments, the DEspR is human DEspR. In some suchembodiments, the DEspR target has a sequence comprising SEQ ID NO:1 oran allelic variant thereof. In some such embodiments of these methods,an antibody or antibody fragment thereof that specifically binds toDEspR and inhibits DEspR biological activity blocks interaction of DEspRwith VEGFsp. In some such embodiments, the VEGFsp has a sequencecomprising the sequence of SEQ ID NO:2. In some such embodiments, theantibody or antibody fragment thereof is specific for an epitope ofDEspR comprising an extracellular portion of DEspR. In some embodiments,the antibody or antibody fragment thereof is specific for an epitope ofDEspR comprising amino acids 1-9 of SEQ ID NO:1.

In some such embodiments of these compositions and methods forinhibiting angiogenesis, the anti-DEspR antibody or antibody-fragmentthereof is the anti-DEspR 7C5B2 antibody or fragment thereof. In somesuch embodiments, the anti-DEspR antibody or antibody-fragment thereofcomprises one or more heavy chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQID NO: 7. In some such embodiments, the anti-DEspR antibody orantibody-fragment thereof comprises one or more light chain CDR regionscomprising a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID NO: 12. In some such embodiments, theanti-DEspR antibody or antibody-fragment thereof comprises one or moreheavy chain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and one ormore light chain CDR regions comprising a sequence selected from thegroup consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. Insome such embodiments, the anti-DEspR antibody or antibody-fragmentthereof comprises a variable heavy (V_(H)) chain amino acid sequenceselected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQID NO: 17. In some such embodiments, the anti-DEspR antibody orantibody-fragment thereof comprises a variable light (V_(L)) chain aminoacid sequence selected from the group consisting of SEQ ID NO: 9, SEQ IDNO: 18, and SEQ ID NO: 19, and fragments thereof.

In other embodiments of these compositions and methods for inhibitingangiogenesis, monoclonal anti-DEspR antibodies or antibody fragmentsthereof that specifically bind to DEspR are provided having one or morebiological characteristics of the 7C5B2 monoclonal antibody. In somesuch embodiments, having a biological characteristic of the 7C5B2monoclonal antibody can include having an ED₅₀ value (i.e., the dosetherapeutically effective in 50% of the population) at or around theED₅₀ value of the 7C5B2 antibody for the given population; or having anEC₅₀ value (i.e., the dose that achieves a half-maximal inhibition of agiven parameter or phenotype) at or around the EC₅₀ value of the 7C5B2antibody for a given parameter or phenotye. For example, in someembodiments of these aspects, the given parameter or phenotype to beinhibited by the antibody that specifically binds to DEspR can include,but is not limited to, the mean total tube number in an in vitrotubulogenesis assay, the mean total tube length in an in vitrotubulogenesis assay, the mean number of branching points in an in vitrotubulogenesis assay, the mean number of vessel connections in an invitro tubulogenesis assay, and tumor cell invasiveness.

In some embodiments these compositions and methods for inhibitingangiogenesis, a humanized anti-DEspR monoclonal antibody or antibodyfragment thereof is provided for use in the compositions and methods forinhibiting angiogenesis as described herein. In some embodiments, one ormore variable heavy chain CDR regions of the humanized anti-DEspRantibody or antibody fragment thereof comprises a sequence selected fromthe group consisting of SEQ ID NO: 5-SEQ ID NO: 7. In some embodiments,one or more variable light chain CDR regions of the humanized anti-DEspRantibody or antibody fragment thereof comprises a sequence selected fromthe group consisting of SEQ ID NO: 10-SEQ ID NO: 12. In some suchembodiments, the anti-DEspR antibody or antibody-fragment thereofcomprises one or more heavy chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQID NO: 7, and one or more light chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, orSEQ ID NO: 12. In some embodiments, the humanized anti-DEspR monoclonalantibody comprises mutated human IgG1 framework regions andantigen-binding complementarity-determining regions (CDRs) selected fromthe group consisting of SEQ ID NO: 5-SEQ ID NO: 7 and the groupconsisting of SEQ ID NO: 10-SEQ ID NO: 12, that blocks binding of humanDEspR to its ligands. In some embodiments, the humanized anti-DEspRantibody comprises mutated human IgG4 framework regions andantigen-binding complementarity-determining regions (CDRs) from theselected from the group consisting of SEQ ID NO: 5-SEQ ID NO: 7 and thegroup consisting of SEQ ID NO: 10-SEQ ID NO: 12, that blocks binding ofhuman DEspR to its ligands.

In other embodiments of these aspects, the anti-DEspR antibody is anantibody fragment having specificity for the same epitope as themonoclonal anti-DEspR antibody 7C5B2, described herein, and produced byhybridoma 7C5B2. In some such embodiments, the anti-DEspR antibody is anantibody fragment comprising one or more variable heavy chain CDRsequences selected from the group consisting of SEQ ID NO: 5-SEQ ID NO:7 and/or one or more variable light chain CDR sequences selected fromthe the group consisting of SEQ ID NO: 10-SEQ ID NO: 12 of the 7C5B2monoclonal antibody. In some embodiments, the antibody fragment is a Fabfragment. In some embodiments, the anti-DEspR antibody fragment is aFab' fragment. In some embodiments, the anti-DEspR antibody fragment isa Fd fragment. In some embodiments, the anti-DEspR antibody fragment isa Fd′ fragment. In some embodiments, the antibody fragment is a Fvfragment. In some embodiments, the anti-DEspR antibody fragment is a dAbfragment. In some embodiments, the anti-DEspR antibody fragmentcomprises isolated CDR regions. In some embodiments, the anti-DEspRantibody fragment is a F(ab')₂ fragment. In some embodiments, theanti-DEspR antibody fragment is a single chain antibody molecule. Insome embodiments, the anti-DEspR antibody fragment is a diabodycomprising two antigen binding sites. In some embodiments, theanti-DEspR antibody fragment is a linear antibody comprising a pair oftandem Fd segments (V_(H)-C_(H)1-V_(H)-C_(H)1).

Accordingly, in some aspects, the disease or disorder dependent ormodulated by angiogenesis is cancer, where the rapidly dividingneoplastic cancer cells require an efficient blood supply to sustaintheir continual growth of the tumor. Inhibition of angiogenesis or tumorcell invasiveness or a combination thereof using the compositions andtherapeutic methods described herein at the primary tumor site andsecondary tumor site serve to prevent and limit metastasis andprogression of disease.

Accordingly, in some aspects, provided herein are methods to treat asubject having or at risk for a cancer or tumor comprising administeringan effective amount of an anti-DEspR antibody or antibody fragmentthereof. In some such embodiments of these methods for treating cancer,the anti-DEspR antibody or antibody fragment thereof is the anti-DEspR7C5B2 antibody or fragment thereof. In some such embodiments, theanti-DEspR antibody or antibody-fragment thereof comprises one or moreheavy chain CDR regions comprising a sequence selected from the groupconsisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some suchembodiments, the anti-DEspR antibody or antibody-fragment thereofcomprises one or more light chain CDR regions comprising a sequenceselected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, andSEQ ID NO: 12. In some such embodiments, the anti-DEspR antibody orantibody-fragment thereof comprises one or more heavy chain CDR regionscomprising a sequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO: 6, and SEQ ID NO: 7 and one or more light chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some such embodiments,the anti-DEspR antibody or antibody-fragment thereof comprises avariable heavy (V_(H)) chain amino acid sequence selected from the groupconsisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID NO: 17. In some suchembodiments, the anti-DEspR antibody or antibody-fragment thereofcomprises a variable light (V_(L)) chain amino acid sequence selectedfrom the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO:19, and fragments thereof.

In some embodiments, the methods can further comprise first selecting ordiagnosing the subject having or at risk for a cancer or tumor. In somesuch embodiments, the diagnosis of the subject can compriseadministering to the subject an anti-DEspR antibody or antibody fragmentthereof coupled to a label, for example, a radioactive label, or a labelused for molecular imaging, as described elsewhere herein. In suchembodiments, detection of the labeled anti-DEspR antibody or antibodyfragment is indicative of the subject having a cancer or tumor.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Included in this definition are benign andmalignant cancers, as well as dormant tumors or micrometastases.Accordingly, the terms “cancer” or “tumor” as used herein refers to anuncontrolled growth of cells which interferes with the normalfunctioning of the bodily organs and systems, including cancer stemcells and tumor vascular niches. A subject that has a cancer or a tumoris a subject having objectively measurable cancer cells present in thesubject's body. Included in this definition are benign and malignantcancers, as well as dormant tumors or micrometastases. Cancers whichmigrate from their original location and seed vital organs caneventually lead to the death of the subject through the functionaldeterioration of the affected organs. Hematopoietic cancers, such asleukemia, are able to out-compete the normal hematopoietic compartmentsin a subject, thereby leading to hematopoietic failure (in the form ofanemia, thrombocytopenia and neutropenia) ultimately causing death.

By “metastasis” is meant the spread of cancer from its primary site toother places in the body. Cancer cells can break away from a primarytumor, penetrate into lymphatic and blood vessels, circulate through thebloodstream, and grow in a distant focus (metastasize) in normal tissueselsewhere in the body. Metastasis can be local or distant. Metastasis isa sequential process, contingent on tumor cells breaking off from theprimary tumor, traveling through the bloodstream, and stopping at adistant site. At the new site, the cells establish a blood supply andcan grow to form a life-threatening mass. Both stimulatory andinhibitory molecular pathways within the tumor cell regulate thisbehavior, and interactions between the tumor cell and host cells in thedistant site are also significant.

Metastases are most often detected through the sole or combined use ofmagnetic resonance imaging (MRI) scans, computed tomography (CT) scans,blood and platelet counts, liver function studies, chest X-rays and bonescans in addition to the monitoring of specific symptoms.

Examples of cancer include but are not limited to, carcinoma, lymphoma,blastoma, sarcoma, and leukemia. More particular examples of suchcancers include, but are not limited to, basal cell carcinoma, biliarytract cancer; bladder cancer; bone cancer; brain and CNS cancer; breastcancer; cancer of the peritoneum; cervical cancer; choriocarcinoma;colon and rectum cancer; connective tissue cancer; cancer of thedigestive system; endometrial cancer; esophageal cancer; eye cancer;cancer of the head and neck; gastric cancer (including gastrointestinalcancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelialneoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer;lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, and squamous carcinoma of the lung);lymphoma including Hodgkin's and non-Hodgkin's lymphoma; melanoma;myeloma; neuroblastoma; glioblastoma; oral cavity cancer (e.g., lip,tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostatecancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; cancer of therespiratory system; salivary gland carcinoma; sarcoma; skin cancer;squamous cell cancer; stomach cancer; testicular cancer; thyroid cancer;uterine or endometrial cancer; cancer of the urinary system; vulvalcancer; as well as other carcinomas and sarcomas; as well as B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); Hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.

In other aspects, the compositions and methods described herein are usedin the treatment or inhibition or imaging of artherosclerotic plaquesand atherosclerosis. Atherosclerosis is the most common form of vasculardisease and is a disorder of large arteries that underlies most coronaryartery disease, aortic aneurysm, cerebrovascular disease and arterialdisease of lower extremities (Libby, in “The Principles of InternalMedicine”, 15th ed., Braunward et al. (editors), Saunders, Philadelphia,Pa., 2001, pp. 1377-1382.). The pathogenesis of atherosclerosis occursas a reaction to injury (Libby, in “The Principles of InternalMedicine”, 15th ed., Braunward et al. (editors), Saunders, Philadelphia,Pa., 2001, pp. 1377-1382.). The injury to the endothelium can be subtle,resulting in a loss of the ability of the cells to function normally.Examples of types of injury to the endothelium includehypercholesterolemia and mechanical stress (Ross, 1999, N. Engl. J.Med., 340:115).

The process of atherosclerosis involves inflammation, and white bloodcells (e.g., lymphocytes, monocytes, and macrophages) are often presentthroughout the development of atherosclerosis. Atherosclerosis beginswhen monocytes are activated and move out of the bloodstream into thewall of an artery. There, they are transformed into foam cells, whichcollect cholesterol and other fatty materials. In time, these fat-ladenfoam cells accumulate and form atheromas in the lining of the artery'swall, causing a thickening and hardening of the wall. Atheromas can bescattered throughout medium-sized and large arteries, but usually formwhere the arteries branch. Treatment of and diagnosis of atherosclerosisis important because it often leads to heart disease and can also causestroke or other vascular problems such as claudication.

Accordingly, in some embodiments of the aspects described herein,pathological angiogenesis in atherosclerotic plaques and in the vasavasorum of atherosclerotic arteries (coronary and carotid arterydisease) is considered a risk and/or causal factor for vulnerable plaqueprogression and disruption. Thus, in some such embodiments, a subjecthaving an angiogenic disorder to be treated using the compositions andmethods described herein can have or be at risk for atherosclerosis. Asused herein, “atherosclerosis” refers to a disease of the arterial bloodvessels resulting in the hardening of arteries caused by the formationof multiple atheromatous plaques within the arteries. Atherosclerosiscan be associated with other disease conditions, including but notlimited to, coronary heart disease events, cerebrovascular events, acutecoronary syndrome, and intermittent claudication. For example,atherosclerosis of the coronary arteries commonly causes coronary arterydisease, myocardial infarction, coronary thrombosis, and anginapectoris. Atherosclerosis of the arteries supplying the central nervoussystem frequently provokes strokes and transient cerebral ischemia. Inthe peripheral circulation, atherosclerosis causes intermittentclaudication and gangrene and can jeopardize limb viability.Atherosclerosis of an artery of the splanchnic circulation can causemesenteric ischemia. Atherosclerosis can also affect the kidneysdirectly (e.g., renal artery stenosis). Also, persons who havepreviously experienced one or more non-fatal atherosclerotic diseaseevents are those for whom the potential for recurrence of such an eventexists.

Sometimes these other diseases can be caused by or associated with otherthan atherosclerosis. Therefore, in some embodiments, one firstdiagnoses that atherosclerosis is present prior to administering thecompositions described herein to the subject. A subject is “diagnosedwith atherosclerosis ” or “selected as having atherosclerosis” if atleast one of the markers of symptoms of atherosclerosis is present. Inone such embodiment, the subject is “selected” if the person has afamily history of atherosclerosis or carries a known genetic mutation orpolymorphism for high cholesterol. In one embodiment, a subject isdiagnosed by measuring an increase level of C-reactive protein (CRP) inthe absence of other inflammatory disorders. In other embodiments,atherosclerosis is diagnosed by measuring serum levels of homocysteine,fibrinogen, lipoprotein (a), or small LDL particles. Alternatively acomputed tomography scan, which measures calcium levels in the coronaryarteries, can be used to select a subject having atherosclerosis. In oneembodiment, atherosclerosis is diagnosed by an increase in inflammatorycytokines. In one embodiment, increased interleukin-6 levels is used asan indicator to select an individual having atherosclerosis. In otherembodiments, increased interleukin-8 and/or interleukin-17 level is usedas an indicator to select an individual having atherosclerosis.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in age-relatedmacular degeneration. It is known, for example, that VEGF contributes toabnormal blood vessel growth from the choroid layer of the eye into theretina, similar to what occurs during the wet or neovascular form ofage-related macular degeneration. Macular degeneration, often called AMDor ARMD (age-related macular degeneration), is the leading cause ofvision loss and blindness in Americans aged 65 and older. New bloodvessels grow (neovascularization) beneath the retina and leak blood andfluid. This leakage causes permanent damage to light-sensitive retinalcells, which die off and create blind spots in central vision or themacula. Accordingly, encompassed in the methods disclosed herein aresubjects treated for age-related macular degeneration withanti-angiogenic therapy.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in a subject havingdiabetic retinopathy, where abnormal blood vessel growth is associatedwith diabetic eye diseases and diabetic macular edema. When normal bloodvessels in the retina are damaged by tiny blood clots due to diabetes, achain reaction is ignited that culminates in new blood vessel growth.However, the backup blood vessels are faulty; they leak (causing edema),bleed and encourage scar tissue that detaches the retina, resulting insevere loss of vision. Such growth is the hallmark of diabeticretinopathy, the leading cause of blindness among young people indeveloped countries. Therefore, encompassed in the methods disclosedherein are subjects treated for diabetic retinopathy and/or diabeticmacular edema.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in a subject havingrheumatoid arthritis. Rheumatoid arthritis (RA) is characterized bysynovial tissue swelling, leukocyte ingress and angiogenesis, or newblood vessel growth. The expansion of the synovial lining of joints inrheumatoid arthritis (RA) and the subsequent invasion by the pannus ofunderlying cartilage and bone necessitate an increase in the vascularsupply to the synovium, to cope with the increased requirement foroxygen and nutrients. Angiogenesis is now recognized as a key event inthe formation and maintenance of the pannus in RA (Paleolog, E. M.,Arthritis Res. 2002;4 Suppl 3:S81-90; Afuwape A O, Histol Histopathol.2002;17(3):961-72). Even in early RA, some of the earliest histologicalobservations are blood vessels. A mononuclear infiltrate characterizesthe synovial tissue along with a luxuriant vasculature. Angiogenesis isintegral to formation of the inflammatory pannus and withoutangiogenesis, leukocyte ingress could not occur (Koch, A. E., Ann.Rheum. Dis. 2000, 59 Suppl 1:i65-71). Disruption of the formation of newblood vessels would not only prevent delivery of nutrients to theinflammatory site, it could also reduce joint swelling due to theadditional activity of VEGF, a potent proangiogenic factor in RA, as avascular permeability factor. Anti-VEGF hexapeptide RRKRRR (dRK6) cansuppress and mitigate the arthritis severity (Seung-Ah Yoo, et. al.,2005, supra). Accordingly, encompassed in the methods disclosed hereinare subjects having or being treated for rheumatoid arthritis.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in Alzheimer'sdisease Alzheimer's disease (AD) is the most common cause of dementiaworldwide. AD is characterized by an excessive cerebral amyloiddeposition leading to degeneration of neurons and eventually todementia. The exact cause of AD is still unknown. It has been shown byepidemiological studies that long-term use of non-steroidalanti-inflammatory drugs, statins, histamine H2-receptor blockers, orcalcium-channel blockers, all of which are cardiovascular drugs with ananti-angiogenic effects, seem to prevent Alzheimer's disease and/orinfluence the outcome of AD patients. Therefore, AD angiogenesis in thebrain vasculature can play an important role in AD. In Alzheimer'sdisease, the brain endothelium secretes the precursor substrate for thebeta-amyloid plaque and a neurotoxic peptide that selectively killscortical neurons. Moreover, amyloid deposition in the vasculature leadsto endothelial cell apoptosis and endothelial cell activation whichleads to neovascularization. Vessel formation could be blocked by theVEGF antagonist SU 4312 as well as by statins, indicating thatanti-angiogenesis strategies can interfere with endothelial cellactivation in AD (Schultheiss C., el. al., 2006; Grammas P., et. al.,1999) and can be used for preventing and/or treating AD. Accordingly,encompassed in the methods disclosed herein are subjects being treatedfor Alzheimer's disease.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in ischemic regionsin the brain, which can contribute to edema, leaky neovessels, andpredispose a subject to hemorrhagic transformation after an ischemicstroke event, thus worsening the morbidity and mortality risk from thestroke event Inhibition of leaky angiogenic neovessels using thecompositions and methods described herein can reduce neurologic deficitsfrom an ischemic stroke event, as well as prevent the progression tohemorrhagic stroke. Currently, there is no therapy for ischemichemorrhagic transformation nor effective therapies to reduce theneurologic deficits from stroke.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in obesity.Adipogenesis in obesity involves interplay between differentiatingadipocytes, stromal cells, and blood vessels. Close spatial and temporalinterrelationships between blood vessel formation and adipogenesis, andthe sprouting of new blood vessels from preexisting vasculature wascoupled to adipocyte differentiation. Adipogenic/angiogenic cellclusters can morphologically and immunohistochemically be distinguishedfrom crown-like structures frequently seen in the late stages of adiposetissue obesity. Administration of anti-vascular endothelial growthfactor (VEGF) antibodies inhibited not only angiogenesis but also theformation of adipogenic/angiogenic cell clusters, indicating that thecoupling of adipogenesis and angiogenesis is essential fordifferentiation of adipocytes in obesity and that VEGF is a key mediatorof that process. (Satoshi Nishimura et. al., 2007, Diabetes56:1517-1526). It has been shown that the angiogenesis inhibitor,TNP-470 was able to prevent diet-induced and genetic obesity in mice(Ebba Bräkenhielm et. al., Circulation Research, 2004;94:1579). TNP-470reduced vascularity in the adipose tissue, thereby inhibiting the rateof growth of the adipose tissue and obesity development. Accordingly,encompassed in the methods disclosed herein are subjects suffering fromobesity.

In other aspects, the compositions and methods described herein are usedin blocking or inhibiting angiogenesis that occurs in endometriosis.Excessive endometrial angiogenesis is proposed as an important mechanismin the pathogenesis of endometriosis (Healy, D L., et. al., Hum ReprodUpdate. 1998 September-October; 4(5):736-40). The endometrium ofpatients with endometriosis shows enhanced endothelial cellproliferation. Moreover there is an elevated expression of the celladhesion molecule integrin vβ3 in more blood vessels in the endometriumof women with endometriosis when compared with normal women. The U.S.Pat. No. 6,121,230 described the use of anti-VEGF agents in thetreatment of endometriosis and is Patent is incorporated herebyreference. Accordingly, encompassed in the methods disclosed herein aresubjects having or being treated for endometriosis.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

The individual or subject to be treated as described herein in variousembodiments is desirably a human patient, although it is to beunderstood that the methods are effective with respect to all mammals,which are intended to be included in the term “patient” or “subject”. Inthis context, a mammal is understood to include any mammalian species inwhich treatment of diseases associated with angiogenesis is desirable.The terms “subject” and “individual” are used interchangeably herein,and refer to an animal, for example a human, recipient of theDEspR-specific antibodies and antibody fragments described herein. Fortreatment of disease states which are specific for a specific animalsuch as a human subject, the term “subject” refers to that specificanimal. The terms “non-human animals” and “non-human mammals” are usedinterchangeably herein, and include mammals such as rats, mice, rabbits,sheep, cats, dogs, cows, pigs, and non-human primates. The term“subject” also encompasses any vertebrate including but not limited tomammals, reptiles, amphibians and fish. However, advantageously, thesubject is a mammal such as a human, or other mammals such as adomesticated mammal, e.g. dog, cat, horse, and the like, or productionmammal, e.g. cow, sheep, pig, and the like are also encompassed in theterm subject.

Modes of Administration

The DEspR-specific antagonist agents, such as anti-DEspR antibodies orantibody fragments thereof, described herein can be administered to asubject in need thereof by any appropriate route which results in aneffective treatment in the subject. As used herein, the terms“administering,” and “introducing” are used interchangeably and refer tothe placement of an anti-DEspR antibody or antibody fragment thereofinto a subject by a method or route which results in at least partiallocalization of such agents at a desired site, such as a site ofinflammation or cancer, such that a desired effect(s) is produced.

In some embodiments, the anti-DEspR antibody or antibody fragmentthereof is administered to a subject having an angiogenic disorder to beinhibited by any mode of administration that delivers the agentsystemically or to a desired surface or target, and can include, but isnot limited to, injection, infusion, instillation, and inhalationadministration. To the extent that anti-DEspR antibodies or antibodyfragments thereof can be protected from inactivation in the gut, oraladministration forms are also contemplated. “Injection” includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments, the anti-DEspR antibodies or antibody fragmentsthereof for use in the methods described herein are administered byintravenous infusion or injection.

The phrases “parenteral administration” and “administered parenterally”as used herein, refer to modes of administration other than enteral andtopical administration, usually by injection. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” as used herein refer tothe administration of the bispecific or multispecific polypeptide agentother than directly into a target site, tissue, or organ, such as atumor site, such that it enters the subject's circulatory system and,thus, is subject to metabolism and other like processes.

The DEspR-specific antagonists described herein are administered to asubject, e.g., a human subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Local administration, for example, to atumor or cancer site where angiogenesis is occurring, is particularlydesired if extensive side effects or toxicity is associated with the useof the DEspR antagonist. An ex vivo strategy can also be used fortherapeutic applications in some embodiments. Ex vivo strategies involvetransfecting or transducing cells obtained from a subject with apolynucleotide encoding a DEspR antagonist. The transfected ortransduced cells are then returned to the subject. The cells can be anyof a wide range of types including, without limitation, hematopoieticcells (e.g., bone marrow cells, macrophages, monocytes, dendritic cells,T cells, or B cells), fibroblasts, epithelial cells, endothelial cells,keratinocytes, or muscle cells.

In some embodiments, when the DEspR-specific antagonist is an anti-DEspRantibody or antibody fragment thereof, the antibody or antibody fragmentthereof is administered by any suitable means, including parenteral,subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, ifdesired for local immunosuppressive treatment, intralesionaladministration. Parenteral infusions include intramuscular, intravenous,intraarterial, intraperitoneal, or subcutaneous administration. In someembodiments, the antibody or antibody fragment thereof is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic.

In some embodiments, the DEspR-specific antagonist compound isadministered locally, e.g., by direct injections, when the disorder orlocation of the tumor permits, and the injections can be repeatedperiodically. The DEspR-specific antagonist can also be deliveredsystemically to the subject or directly to the tumor cells, e.g., to atumor or a tumor bed following surgical excision of the tumor, in orderto prevent or reduce local recurrence or metastasis, for example of adormant tumor or micrometastases.

Administration by Sonoporation

Antibody-targeted sonoporation methods are contemplated for use in someembodiments of the methods for inhibiting angiogenesis described herein,in order to enhance the efficacy and potency of the therapeuticcompositions comprising anti-DEspR antibodies and antibody fragmentsthereof provided herein.

The inventors have discovered that DEspR-targeted sonoporation ofpharmaceutical compositions comprising anti-DEspR monoclonal antibodiesand antibody fragments provides surprisingly enhanced reduction of tumorgrowth and metastases, indicating enhanced penetration and delivery ofthe compositions, and enhances delivery to sites of pathologicalangiogenesis, and to tumor cells, and tumor initiating cells or cancerstem cells or cancer stem-like cells. Further, the inventors havediscovered that sonoporation of anti-DEspR antibodies and antibodyfragments thereof in combination with other therapeutic agents, such assmall molecule compounds or other drug compounds, can be used to enhancedelivery of the other therapeutic agents, thus providing a means oftargeted and enhanced delivery.

Accordingly, in some embodiments of the methods of inhibitingangiogenesis described herein, anti-DEspR antibodies and antibodyfragments thereof are administered to a subject in need thereof bysonoporation.

As used herein, “sonoporation” refers to the use of sound, preferably atultrasonic frequencies, or the interaction of ultrasound with contrastagents (e.g., stabilized microbubbles) for temporarily modifying thepermeability of cell plasma membranes, thus allowing uptake of largemolecules, such as therapeutic agents. The membrane permeability causedby the sonoporation is transient, leaving the agents trapped inside thecell after the ultrasound exposure. Sonoporation employs acousticcavitation of microbubbles to enhance delivery of large molecules.

Accordingly, in some embodiments of the methods, therapeutic anti-DEspRagents, such as the anti-DEspR antibodies and antibody fragments thereofdescribed herein, mixed with ultrasound contrast agents, such asmicrobubbles, can be injected locally or systemically into a subject inneed of treatment for an angiogenic disorder, and ultrasound can becoupled and even focused into the defined area, e.g., tumor site, toachieve targeted delivery of the anti-DEspR antibodies and antibodyfragments thereof described herein. Additionally, the anti-DEspRantibody or antibody fragment thereof is known to target the tumorvessel endothelium, thus directing the sonoporation to areas ofincreased DEspR expression in tumor endothelial cells. In addition tothe operator-determined focused ultrasound, anti-DEspR targeting of amicrobubble can be used to target the sonoporation-mediated enhancedentry of any therapeutic agent, including antiDEspR monoclonal antibodyper se, into said targeted cancerous areas.

In some embodiments, the methods use focused ultrasound methods toachieve targeted delivery of the anti-DEspR antibodies and antibodyfragments thereof described herein. As used herein, HIFU or “HighIntensity Focused Ultrasound” refers to a non-invasive therapeuticmethod using high-intensity ultrasound to heat and destroy malignant orpathogenic tissue without causing damage to overlying or surroundinghealth tissue. Typically, HIFU has been used in tissue ablationtechniques, whereby the biological effects of HIFU treatment, includingcoagulative necrosis and structural disruption, can be induced in atissue requiring ablation, such as a solid tumor site. However, asdescribed in Khaibullina A. et al., J Nucl Med. 2008 February;49(2):295-302, and WO2010127369, the contents of which are hereinincorporated in their entireties by reference, HIFU can also be used asa means of delivery of therapeutic agents, such as antibodies orantibody fragments thereof.

Methods using contrast-enhanced ultrasound (CEUS) are also contemplatedfor use with anti-DEspR inhibiting agents described herein.Contrast-enhanced ultrasound (CEUS) refers to the application ofultrasound contrast medium and ultrasound contrast agents to traditionalmedical sonography. Ultrasound contrast agents refer to agents that relyon the different ways in which sound waves are reflected from interfacesbetween substances. This can be the surface of a small air bubble or amore complex structure. Commercially available contrast media includegas-filled microbubbles that are administered intravenously to thesystemic circulation. Microbubbles have a high degree of echogenicity,i.e., the ability of an object to reflect the ultrasound waves. Theechogenicity difference between the gas in the microbubbles and the softtissue surroundings of the body is immense, and enhances the ultrasoundbackscatter, or reflection of the ultrasound waves, to produce a uniquesonogram with increased contrast due to the high echogenicitydifference. Contrast-enhanced ultrasound can be used with thecompositions and methods described herein to image a variety ofconditions and disorders, such as angiogenesis dependent disorders, asdescribed herein

A variety of microbubble contrast agents are available for use with thecompositions and methods described herein. Microbubbles can differ intheir shell makeup, gas core makeup, and whether or not they aretargeted.

The microbubble shell material determines how easily the microbubble istaken up by the immune system. A more hydrophilic shell material tendsto be taken up more easily, which reduces the microbubble residence timein the circulation. This reduces the time available for contrastimaging. The shell material also affects microbubble mechanicalelasticity. The more elastic the material, the more acoustic energy itcan withstand before bursting. Example of materials used in currentmicrobubble shells include albumin, galactose, lipid, and polymers, asdescribed in Lindner, J. R. 2004. Microbubbles in medical imaging:current applications and future directions. Nat Rev Drug Discov. 3:527-32, the contents of which are herein incoporated by reference intheir entireties.

The microbubble gas core is an important part of the ultrasound contrastmicrobubble because it determines the echogenicity. When gas bubbles arecaught in an ultrasonic frequency field, they compress, oscillate, andreflect a characteristic echo—this generates the strong and uniquesonogram in contrast-enhanced ultrasound. Gas cores can be composed of,for example, air, or heavy gases like perfluorocarbon, or nitrogen.Heavy gases are less water-soluble so they are less likely to leak outfrom the microbubble to impair echogenicity. Therefore, microbubbleswith heavy gas cores are likely to last longer in circulation.

Regardless of the shell or gas core composition, microbubble size aretypically fairly uniform. They can lie within in a range of 1-4micrometres in diameter. That makes them smaller than red blood cells,which allows them to flow easily through the circulation as well as themicrocirculation.

Targeting ligands that bind to receptors characteristic of angiogenicdisorders, such as DEspR, can be conjugated to microbubbles, enablingthe microbubble complex to accumulate selectively in areas of interest,such as diseased or abnormal tissues. This form of molecular imaging,known as targeted contrast-enhanced ultrasound, will only generate astrong ultrasound signal if targeted microbubbles bind in the area ofinterest. Targeted contrast-enhanced ultrasound has many applications inboth medical diagnostics and medical therapeutics. Microbubbles targetedwith an agent that binds to DEspR, such as an anti-DEspR antibody orantibody fragment thereof, are injected systemically in a small bolus.These DEspR-targeted microbubbles can travel through the circulatorysystem, eventually finding their respective targets and bindingspecifically. Ultrasound waves can then be directed on the area ofinterest. If a sufficient number of DEspR-targeted microbubbles havebound in the area, their compressible gas cores oscillate in response tothe high frequency sonic energy field. The DEspR-targeted microbubblesalso reflect a unique echo that is in stark contrast to the surroundingtissue due to the orders of magnitude mismatch between microbubble andtissue echogenicity. The ultrasound system converts the strongechogenicity into a contrast-enhanced image of the area of interest,revealing the location of the bound DEspR-targeted microbubbles.Detection of bound microbubbles can then show that the area of interestis expressing DEspR, which can be indicative of a certain disease state,or identify particular cells in the area of interest. In addition,targeted sonoporation can be done at the site where DEspR-targetedmicrobubbles are attached, thus achieving targeted delivery of anytherapeutic agent (drug, siRNA, DNA, small molecule) encapsulated in orcarried on the echogenic microbubble.

Accordingly, in some embodiments of the methods described herein, ananti-DEspR antibody or antibody fragment thereof, such as, for example,an anti-DEspR 7C5B2 antibody or fragment thereof, an anti-DEspR antibodyor antibody-fragment thereof comprising one or more heavy chain CDRregions comprising a sequence selected from the group consisting of SEQID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; an anti-DEspR antibody orantibody-fragment thereof comprising one or more light chain CDR regionscomprising a sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO: 11, or SEQ ID NO: 12; an anti-DEspR antibody orantibody-fragment comprising a variable heavy (V_(H)) chain amino acidsequence selected from the group consisting of SEQ ID NO: 4 and SEQ IDNO: 13-SEQ ID NO: 17; and/or an anti-DEspR antibody or antibody-fragmentthereof comprising a variable light (V_(L)) chain amino acid sequenceselected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, andSEQ ID NO: 19, is administered to a subject in need of treatment for anangiogenic disorder, such as for example, cancer, using a targetedultrasound delivery. In some such embodiments, the targeted ultrasounddelivery comprises using microbubbles as contrast agents to which ananti-DEspR antibody or antibody fragment thereof,. In some suchembodiments, the targeted ultrasound is HIFU.

Pharmaceutical Formulations

For the clinical use of the methods described herein, administration ofthe DEspR antagonists, such as the anti-DEspR antibodies or antibodyfragments thereof described herein, can include formulation intopharmaceutical compositions or pharmaceutical formulations forparenteral administration, e.g., intravenous; mucosal, e.g., intranasal;ocular, or other mode of administration. In some embodiments, the antiDEspR antibodies or antibody fragments thereof described herein can beadministered along with any pharmaceutically acceptable carriercompound, material, or composition which results in an effectivetreatment in the subject. Thus, a pharmaceutical formulation for use inthe methods described herein can contain an anti-DEspR antibody orantibody fragment thereof as described herein in combination with one ormore pharmaceutically acceptable ingredients.

The phrase “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio. The phrase “pharmaceutically acceptablecarrier” as used herein means a pharmaceutically acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, solvent, media, encapsulating material, manufacturing aid(e.g., lubricant, talc magnesium, calcium or zinc stearate, or stericacid), or solvent encapsulating material, involved in maintaining thestability, solubility, or activity of, an anti-DEspR antibody orantibody fragment thereof. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. The terms “excipient”, “carrier”,“pharmaceutically acceptable carrier” or the like are usedinterchangeably herein.

The anti-DEspR antibodies or antibody fragments thereof described hereincan be specially formulated for administration of the compound to asubject in solid, liquid or gel form, including those adapted for thefollowing: (1) parenteral administration, for example, by subcutaneous,intramuscular, intravenous or epidural injection as, for example, asterile solution or suspension, or sustained-release formulation; (2)topical application, for example, as a cream, ointment, or acontrolled-release patch or spray applied to the skin; (3)intravaginally or intrarectally, for example, as a pessary, cream orfoam; (4) ocularly; (5) transdermally; (6) transmucosally; or (79)nasally. Additionally, an anti-DEspR antibody or antibody fragmentthereof can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. Nos. 3,773,919; and 35 3,270,960.

Therapeutic formulations of the DEspR-specific antagonist agents, suchas anti-DEspR antibodies or antibody fragments thereof, described hereincan be prepared for storage by mixing a DEspR-specific antagonist havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980)), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG). Exemplary lyophilized anti-VEGF antibodyformulations are described in WO 97/04801, expressly incorporated hereinbe reference.

Optionally, but preferably, the formulations comprising the compositionsdescribed herein contain a pharmaceutically acceptable salt, typically,e.g., sodium chloride, and preferably at about physiologicalconcentrations. Optionally, the formulations of the invention cancontain a pharmaceutically acceptable preservative. In some embodimentsthe preservative concentration ranges from 0.1 to 2.0%, typically v/v.Suitable preservatives include those known in the pharmaceutical arts.Benzyl alcohol, phenol, m-cresol, methylparaben, and propylparaben areexamples of preservatives. Optionally, the formulations of the inventioncan include a pharmaceutically acceptable surfactant at a concentrationof 0.005 to 0.02%.

The therapeutic formulations of the compositions comprisingDEspR-specific antagonists, such as anti-DEspR antibodies and antibodyfragments thereof, described herein can also contain more than oneactive compound as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. For example, in some embodiments, it can bedesirable to further provide antibodies which bind to EGFR, VEGF (e.g.an antibody which binds a different epitope on VEGF), VEGFR, or ErbB2(e.g., Herceptin™) Alternatively, or in addition, the composition cancomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orVEGFR antagonist. Such molecules are suitably present in combination inamounts that are effective for the purpose intended.

The active ingredients of the therapeutic formulations of thecompositions comprising DEspR-specific antagonists described herein canalso be entrapped in microcapsules prepared, for example, bycoacervation techniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

In some embodiments, sustained-release preparations can be used.Suitable examples of sustained-release preparations includesemipermeable matrices of solid hydrophobic polymers containing theDEspR-specific antagonist, such as an anti-DEspR antibody, in which thematrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and y ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated antibodies remain in the body for a longtime, they can denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization can beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

The therapeutic formulations to be used for in vivo administration, suchas parenteral administration, in the methods described herein can besterile, which is readily accomplished by filtration through sterilefiltration membranes, or other methods known to those of skill in theart.

Dosages and Duration

The DEspR-specific antagonists described herein, such as anti-DEspRantibodies and antibody fragments thereof, are formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular subject being treated, the clinical condition ofthe individual subject, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. The“therapeutically effective amount” of the DEspR-specific antagonist tobe administered will be governed by such considerations, and refers tothe minimum amount necessary to ameliorate, treat, or stabilize, thecancer; to increase the time until progression (duration of progressionfree survival) or to treat or prevent the occurrence or recurrence of atumor, a dormant tumor, or a micrometastases. The DEspR -specificantagonist is optionally formulated with one or more additionaltherapeutic agents currently used to prevent or treat cancer or a riskof developing a cancer. The effective amount of such other agentsdepends on the amount of VEGF-specific antagonist present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as used herein before or about from 1 to 99% ofthe heretofore employed dosages.

Depending on the type and severity of the disease, about lμg/kg to 100mg/kg (e.g., 0.1-20 mg/kg) of a DEspR-specific antagonist is an initialcandidate dosage for administration to a subject, whether, for example,by one or more separate administrations, or by continuous infusion. Atypical daily dosage might range from about 1μg/kg to about 100 mg/kg ormore, depending on the factors mentioned above. Particularly desirabledosages include, for example, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, and 15mg/kg. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until, forexample, the cancer is treated, as measured by the methods describedabove or known in the art. However, other dosage regimens can be useful.In one non-limiting example, if the DEspR-specific antagonist is ananti-DEspR antibody or antibody fragment thereof, the anti-DEspRantibody or antibody fragment thereof is administered once every week,every two weeks, or every three weeks, at a dose range from about 5mg/kg to about 15 mg/kg, including but not limited to 5 mg/kg, 7.5mg/kg, 10 mg/kg or 15 mg/kg. The progress of using the methods describedherein can be easily monitored by conventional techniques and assays.

The duration of a therapy using the methods described herein willcontinue for as long as medically indicated or until a desiredtherapeutic effect (e.g., those described herein) is achieved. Incertain embodiments, the DEspR-specific antagonist therapy, such as aDEspR-specific antibody or antibody fragment described herein iscontinued for 1 month, 2 months, 4 months, 6 months, 8 months, 10months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years,or for a period of years up to the lifetime of the subject.

Efficacy of the Treatment

The efficacy of the treatment methods for cancer comprising therapeuticformulations of the compositions comprising the DEspR-specificantagonists described herein can be measured by various endpointscommonly used in evaluating cancer treatments, including but not limitedto, tumor regression, tumor weight or size shrinkage, time toprogression, duration of survival, progression free survival, overallresponse rate, duration of response, and quality of life. Because theDEspR-specific antagonists, e.g., anti-DEspR antibodies and antibodyfragments thereof, described herein target the tumor vasculature, cancercells, and some cancer stem cell subsets, they represent a unique classof multi-targeting anticancer drugs, and therefore can require uniquemeasures and definitions of clinical responses to drugs. For example,tumor shrinkage of greater than 50% in a 2-dimensional analysis is thestandard cut-off for declaring a response. However, theanti-DEspR-antibodies or antibody fragments thereof described herein cancause inhibition of metastatic spread without shrinkage of the primarytumor, or can simply exert a tumoristatic effect. Accordingly, novelapproaches to determining efficacy of an anti-angiogenic therapy shouldbe employed, including for example, measurement of plasma or urinarymarkers of angiogenesis, and measurement of response through molecularimaging, using, for example, an DEspR-antibody or antibody fragmentconjugated to a label, such as a microbubble. In the case of cancers,the therapeutically effective amount of the DEspR-antibody or antibodyfragment thereof can reduce the number of cancer cells; reduce the tumorsize; inhibit (i.e., slow to some extent and preferably stop) cancercell infiltration into peripheral organs; inhibit (i.e., slow to someextent and preferably stop) tumor metastasis; inhibit, to some extent,tumor growth; and/or relieve to some extent one or more of the symptomsassociated with the disorder. To the extent the DEspR-antibody orantibody fragment thereof can prevent growth and/or kill existing cancercells, it can be cytostatic and/or cytotoxic. For cancer therapy,efficacy in vivo can, for example, be measured by assessing the durationof survival, duration of progression free survival (PFS), the responserates (RR), duration of response, and/or quality of life.

In other embodiments, described herein are methods for increasingprogression free survival of a human subject susceptible to or diagnosedwith a cancer. Time to disease progression is defined as the time fromadministration of the drug until disease progression or death. In apreferred embodiment, the combination treatment of the invention using aDEspR-specific antagonist, such as an anti-DEspR antibody or antibodyfragment thereof, and one or more chemotherapeutic agents significantlyincreases progression free survival by at least about 1 month, 1.2months, 2 months, 2.4 months, 2.9 months, 3.5 months, preferably byabout 1 to about 5 months, when compared to a treatment withchemotherapy alone. In another embodiment, the methods decribed hereinsignificantly increase response rates in a group of human subjectssusceptible to or diagnosed with a cancer who are treated with varioustherapeutics. Response rate is defined as the percentage of treatedsubjects who responded to the treatment. In one embodiment, thecombination treatment described herein using a DEspR-specificantagonist, such as an anti-DEspR antibody or antibody fragment thereof,and one or more chemotherapeutic agents significantly increases responserate in the treated subject group compared to the group treated withchemotherapy alone.

As used herein, the terms “treat” “treatment” “treating,” or“amelioration” refer to therapeutic treatments, wherein the object is toreverse, alleviate, ameliorate, inhibit, slow down or stop theprogression or severity of a condition associated with, a disease ordisorder. The term “treating” includes reducing or alleviating at leastone adverse effect or symptom of a condition, disease or disorderassociated with a chronic immune condition, such as, but not limited to,a chronic infection or a cancer. Treatment is generally “effective” ifone or more symptoms or clinical markers are reduced. Alternatively,treatment is “effective” if the progression of a disease is reduced orhalted. That is, “treatment” includes not just the improvement ofsymptoms or markers, but also a cessation of at least slowing ofprogress or worsening of symptoms that would be expected in absence oftreatment. Beneficial or desired clinical results include, but are notlimited to, alleviation of one or more symptom(s), diminishment ofextent of disease, stabilized (i.e., not worsening) state of disease,delay or slowing of disease progression, amelioration or palliation ofthe disease state, and remission (whether partial or total), whetherdetectable or undetectable. The term “treatment” of a disease alsoincludes providing relief from the symptoms or side-effects of thedisease (including palliative treatment).

For example, in some embodiments, the methods described herein compriseadministering an effective amount of the anti-DEspR antibodies orantibody fragments thereof described herein to a subject in order toalleviate a symptom of a cancer, or other such disorder characterized byexcess or unwanted angiogenesis. As used herein, “alleviating a symptomof a cancer” is ameliorating or reducing any condition or symptomassociated with the cancer. As compared with an equivalent untreatedcontrol, such reduction or degree of prevention is at least 5%, 10%,20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standardtechnique. Ideally, the cancer is completely cleared as detected by anystandard method known in the art, in which case the cancer is consideredto have been treated. A patient who is being treated for a cancer is onewho a medical practitioner has diagnosed as having such a condition.Diagnosis can be by any suitable means. Diagnosis and monitoring caninvolve, for example, detecting the level of cancer cells in abiological sample (for example, a tissue or lymph node biopsy, bloodtest, or urine test), detecting the level of a surrogate marker of thecancer in a biological sample, detecting symptoms associated with thespecific cancer, or detecting immune cells involved in the immuneresponse typical of such a cancer infections.

The term “effective amount” as used herein refers to the amount of ananti-DEspR antibody or antibody fragment thereof needed to alleviate atleast one or more symptom of the disease or disorder, and relates to asufficient amount of pharmacological composition to provide the desiredeffect, i.e., inhibit the formation of new blood vessels. The term“therapeutically effective amount” therefore refers to an amount of ananti-DEspR antibody or antibody fragment thereof using the methods asdisclosed herein, that is sufficient to effect a particular effect whenadministered to a typical subject. An effective amount as used hereinwould also include an amount sufficient to delay the development of asymptom of the disease, alter the course of a symptom disease (forexample but not limited to, slow the progression of a symptom of thedisease), or reverse a symptom of the disease. Thus, it is not possibleto specify the exact “effective amount”. However, for any given case, anappropriate “effective amount” can be determined by one of ordinaryskill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dosage can vary depending upon the dosage formemployed and the route of administration utilized. The dose ratiobetween toxic and therapeutic effects is the therapeutic index and canbe expressed as the ratio LD₅₀/ED₅₀. Compositions and methods thatexhibit large therapeutic indices are preferred. A therapeuticallyeffective dose can be estimated initially from cell culture assays.Also, a dose can be formulated in animal models to achieve a circulatingplasma concentration range that includes the IC₅₀ (i.e., theconcentration of the anti-DEspR antibody or antibody fragment thereof),which achieves a half-maximal inhibition of symptoms) as determined incell culture, or in an appropriate animal model. Levels in plasma can bemeasured, for example, by high performance liquid chromatography. Theeffects of any particular dosage can be monitored by a suitablebioassay. The dosage can be determined by a physician and adjusted, asnecessary, to suit observed effects of the treatment.

Combination Antiangiogenic Therapies

In other embodiments, the methods provided for inhibiting angiogenesisin a tissue of a subject or individual having a disease or disorderdependent or modulated by angiogenesis by administering to the subject atherapeutically effective amount of a composition comprising anangiogenesis-inhibiting amount of an anti-DEspR inhibitor, such as ananti-DEspR antibody or antibody fragment thereof, can further compriseadministration one or more additional treatments such as angiogenicinhibitors, chemotherapy, radiation, surgery, or other treatments knownto those of skill in the art to inhibit angiogenesis.

In some embodiments, the methods described herein further compriseadministration of a combination of at least one DEspR-specificantagonist, such an anti-DEspR antibody or antibody fragment thereof,with one or more additional anti-cancer therapies. Examples ofadditional anti-cancer therapies include, without limitation, surgery,radiation therapy (radiotherapy), biotherapy, immunotherapy,chemotherapy, or a combination of these therapies. In addition,cytotoxic agents, anti-angiogenic and anti-proliferative agents can beused in combination with the DEspR-specific antagonist.

In certain aspects of any of the methods and uses, the inventionprovides treating cancer by administering effective amounts of ananti-DEspR antibody and one or more chemotherapeutic agents to a subjectsusceptible to, or diagnosed with, locally recurrent or previouslyuntreated cancer. A variety of chemotherapeutic agents can be used inthe combined treatment methods and uses of the invention. An exemplaryand non-limiting list of chemotherapeutic agents contemplated for use inthe methods described herein is provided under “Definition,” ordescribed herein.

In some embodiments, the methods described herein compriseadministration of a DEspR-specific antagonist with one or morechemotherapeutic agents (e.g., a cocktail) or any combination thereof.In certain embodiments, the chemotherapeutic agent is for example,capecitabine, taxane, anthracycline, paclitaxel, docetaxel, paclitaxelprotein-bound particles (e.g., Abraxane™) doxorubicin, epirubicin,5-fluorouracil, cyclophosphamide or combinations thereof therapy. Asused herein, combined administration includes simultaneousadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities. Preparation and dosingschedules for such chemotherapeutic agents can be used according tomanufacturers' instructions or as determined empirically by the skilledpractitioner. Preparation and dosing schedules for chemotherapy are alsodescribed in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins,Baltimore, Md. (1992). Accordingly, in some embodiments, thechemotherapeutic agent can precede, or follow administration of theDEspR-specific antagonist or can be given simultaneously therewith.

In some other embodiments of the methods described herein, othertherapeutic agents useful for combination tumor therapy with the DEspRantagonists, such as antibodies, of the invention include antagonists ofother factors that are involved in tumor growth, such as EGFR, ErbB2(also known as Her2), ErbB3, ErbB4, or TNF. In some embodiments, it canbe beneficial to also administer one or more cytokines to the subject.In some embodiments, the DEspR antagonist is co-administered with agrowth inhibitory agent. For example, the growth inhibitory agent can beadministered first, followed by the DEspR antagonist. However,simultaneous administration or administration of the DEspR antagonistfirst is also contemplated. Suitable dosages for the growth inhibitoryagent are those presently used and can be lowered due to the combinedaction (synergy) of the growth inhibitory agent and DEspR antagonist.

Examples of additional angiogenic inhibitors that can be used incombination with the DEspR inhibitors, such as anti-DEspR antibodies andantibody fragments thereof, described herein include, but are notlimited to: direct angiogenesis inhibitors, Angiostatin, Bevacizumab(Avastin®), Arresten, Canstatin, Combretastatin, Endostatin, NM-3,Thrombospondin, Tumstatin, 2-methoxyestradiol, cetuximab (Erbitux®),panitumumab (Vectibix™), trastuzumab (Herceptin®) and Vitaxin; andindirect angiogenesis inhibitors: ZD1839 (Iressa), ZD6474, 0S1774(Tarceva), CI1033, PKI1666, IMC225 (Erbitux), PTK787, SU6668, SU11248,Herceptin, and IFN-α, CELEBREX® (Celecoxib), THALOMID® (Thalidomide),and IFN-α.

In some embodiments, the additional angiogenesis inhibitors for use inthe methods described herein include but are not limited to smallmolecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenicgrowth factor receptors. The three TKIs that are currently approved asanti-cancer therapies are erlotinib (Tarceva®), sorafenib (Nexavar®),and sunitinib (Sutent®).

In some embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include but are not limited to inhibitors of mTOR(mammalian target of rapamycin) such as temsirolimus (Toricel™),bortezomib (Velcade®), thalidomide (Thalomid®), and Doxycyclin,

In other embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include one or more drugs that target the VEGF pathway.Bevacizumab (Avastin®) was the first drug that targeted new bloodvessels to be approved for use against cancer. It is a monoclonalantibody that binds to VEGF, thereby blocking VEGF from reaching theVEGF receptor (VEGFR). Other drugs, such as sunitinib (Sutent®) andsorafenib (Nexavar®), are small molecules that attach to the VEGFreceptor itself, preventing it from being turned on. Such drugs arecollectively termed VEGF inhibitors. As the VEGF/VPF protein interactswith the VEGFRs, inhibition of either the ligand VEGF, e.g. by reducingthe amount that is available to interact with the receptor; orinhibition of the receptor's intrinsic tyrosine kinase activity, blocksthe function of this pathway. This pathway controls endothelial cellgrowth, as well as permeability, and these functions are mediatedthrough the VEGFRs.

Accordingly, as described herein, “VEGF inhibitors” for use asangiogenesis inhibitors include any compound or agent that produces adirect or indirect effect on the signaling pathways that promote growth,proliferation and survival of a cell by inhibiting the function of theVEGF protein, including inhibiting the function of VEGF receptorproteins. These include any organic or inorganic molecule, including,but not limited to modified and unmodified nucleic acids such asantisense nucleic acids, RNAi agents such as siRNA or shRNA, peptides,peptidomimetics, receptors, ligands, and antibodies that inhibit theVEGF signaling pathway. The siRNAs are targeted at components of theVEGF pathways and can inhibit the VEGF pathway. Preferred VEGFinhibitors, include for example, AVASTIN® (bevacizumab), an anti-VEGFmonoclonal antibody of Genentech, Inc. of South San Francisco, Calif.,VEGF Trap (Regeneron/Aventis). Additional VEGF inhibitors includeCP-547,632 (3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride;Pfizer Inc. , NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, &SU6668 (formerly Sugen Inc., now Pfizer, New York, N.Y.), ZD-6474(AstraZeneca), ZD4190 which inhibits VEGF-R2 and -R1 (AstraZeneca),CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC 412 (Novartis), AEE788(Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; BayerPharmaceuticals and Onyx Pharmaceuticals), vatalanib (also known asPTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptaniboctasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862(glufanide disodium, Cytran Inc. of Kirkland, Wash., USA),VEGFR2-selective monoclonal antibody DC101 (ImClone Systems, Inc.),angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) andChiron (Emeryville, Calif.), Sirna-027 (an siRNA-based VEGFR1 inhibitor,Sirna Therapeutics, San Francisco, Calif.) Caplostatin, solubleectodomains of the VEGF receptors, Neovastat (

terna Zentaris Inc; Quebec City, Calif.), ZM323881 CalBiochem. Calif.,USA), pegaptanib (Macugen) (Eyetech Pharmaceuticals), an anti-VEGFaptamer and combinations thereof.

VEGF inhibitors are also disclosed in U.S. Pat. Nos. 6,534,524 and6,235,764, both of which are incorporated in their entirety. AdditionalVEGF inhibitors are described in, for example in WO 99/24440 (publishedMay 20, 1999), International Application PCT/IB99/00797 (filed May 3,1999), in WO 95/21613 (published Aug. 17, 1995), WO 99/61422 (publishedDec. 2, 1999), U.S. Pat. Publ. No. 20060094032 “siRNA agents targetingVEGF”, U.S. Patent 6, 534,524 (discloses AG13736), U.S. Patent 5,834,504(issued Nov. 10, 1998), WO 98/50356 (published Nov. 12, 1998), U.S. Pat.5, 883,113 (issued Mar. 16, 1999), U.S. Pat. 5, 886,020 (issued Mar. 23,1999), U.S. Patent 5,792,783 (issued Aug. 11, 1998), U.S. Pat. No.6,653,308 (issued Nov. 25, 2003), WO 99/10349 (published Mar. 4, 1999),WO 97/32856 (published Sep. 12, 1997), WO 97/22596 (published Jun. 26,1997), WO 98/54093 (published Dec. 3, 1998), WO 98/02438 (published Jan.22, 1998), WO 99/16755 (published Apr. 8, 1999), and WO 98/02437(published Jan. 22, 1998), WO 01/02369 (published Jan. 11, 2001); U.S.Provisional Application No. 60/491,771 piled Jul. 31, 2003); U.S.Provisional Application No. 60/460,695 (filed Apr. 3, 2003); and WO03/106462A1 (published Dec. 24, 2003). Other examples of VEGF inhibitorsare disclosed in International Patent Publications WO 99/62890 publishedDec. 9, 1999, WO 01/95353 published Dec. 13, 2001 and WO 02/44158published Jun. 6, 2002.

In other embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include anti-angiogenic factors such as alpha-2antiplasmin (fragment), angiostatin (plasminogen fragment),antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59complement fragment, endostatin (collagen XVIII fragment), fibronectinfragment, gro-beta (a C-X-C chemokine), heparinases heparinhexasaccharide fragment, human chorionic gonadotropin (hCG), interferonalpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12,kringle 5 (plasminogen fragment), beta-thromboglobulin, EGF (fragment),VEGF inhibitor, endostatin, fibronection (45 kD fragment), highmolecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of HGF,PF-4, serpin proteinase inhibitor 8, TGF-beta-1, thrombospondin-1,prosaposin, p53, angioarrestin, metalloproteinase inhibitors (TIMPs),2-Methoxyestradiol, placental ribonuclease inhibitor, plasminogenactivator inhibitor, prolactin 16 kD fragment, proliferin-relatedprotein (PRP), retinoids, tetrahydrocortisol-S transforming growthfactor-beta (TGF-b), vasculostatin, and vasostatin (calreticulinfragment). pamidronate thalidomide, TNP470, the bisphosphonate familysuch as amino-bisphosphonate zoledronic acid. bombesin/gastrin-releasingpeptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol A M, et.al., British Journal of Cancer (2004) 90, 245-252), anti-VEGF peptideRRKRRR (dRK6) (Seung-Ah Yoo, J. Immuno, 2005, 174: 5846-5855).

Thus, in connection with the administration of a DEspR inhibitor, suchas anti-DEspR antibodies and antibody fragments thereof, a compoundwhich inhibits angiogenesis indicates that administration in aclinically appropriate manner results in a beneficial effect for atleast a statistically significant fraction of patients, such asimprovement of symptoms, a cure, a reduction in disease load, reductionin tumor mass or cell numbers, extension of life, improvement in qualityof life, or other effect generally recognized as positive by medicaldoctors familiar with treating the particular type of disease orcondition.

Examples of additional DEspR inhibitors include, but are not limited to,molecules which block the binding of VEGFsp, ET-1 and/or other ET-1orVEGFsp-like ligands to DEspR, compounds which interfere withdownstream signaling events of DEspR, or other compounds or agents thatinhibit activation of the receptor. Such compounds can bind to DEspR andprevent binding of VEGFsp, ET-1 or other mimetic ligands. Otherinhibitors including small molecules that bind to the DEspR domain thatbinds to VEGFsp, soluble DEspR receptors, peptides containing the DEspRET-1 and/or VEGFsp binding domains, etc. are also contemplated.

The compositions described herein can also contain more than one activecompound as necessary for the particular indication being treated, andthese active compounds are preferably those with complementaryactivities that do not adversely affect each other. For example, it canbe desirable to further provide antibodies or antagonists that bind toEGFR, VEGF, VEGFR, or ErbB2 (e.g., Herceptin.™). Alternatively, or inaddition, the composition can comprise a cytotoxic agent, cytokine,growth inhibitory agent and/or VEGFR antagonist. Such molecules aresuitably present in combination in amounts that are effective for thepurpose intended.

In certain aspects of any of the methods and uses described herein,other therapeutic agents useful for combination cancer therapy with theantibody of the invention include other anti-angiogenic agents. Manyanti-angiogenic agents have been identified and are known in the arts,including those listed by Carmeliet and Jain (2000). In someembodiments, the DEspR antagonist, such as a humanized anti-DEspRantibody or antibody fragment thereof described herein is used incombination with a VEGF antagonist or a VEGF receptor antagonist such asVEGF variants, soluble VEGF receptor fragments, aptamers capable ofblocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low moleculeweight inhibitors of VEGFR tyrosine kinases and any combinationsthereof. Alternatively, or in addition, two or more anti-DEspRantagonists can be co-administered to the subject.

For the treatment of diseases, as described herein, the appropriatedosage of DEspR-specific antagonists will depend on the type of diseaseto be treated, as defined above, the severity and course of the disease,whether the DEspR-specific antagonist is administered for preventive ortherapeutic purposes, previous therapeutic indications, the subject'sclinical history and response to the DEspR-specific antagonist, and thediscretion of the attending physician. The DEspR-specific antagonist issuitably administered to the subject at one time or over a series oftreatments. In a combination therapy regimen, the DEspR-specificantagonist and the one or more anti-cancer therapeutic agents describedherein are administered in a therapeutically effective or synergisticamount. As used herein, a therapeutically effective amount is such thatco-administration of a DEspR-specific antagonist and one or more othertherapeutic agents, or administration of a composition described herein,results in reduction or inhibition of the cancer as described herein. Atherapeutically synergistic amount is that amount of a DEspR -specificantagonist and one or more other therapeutic agents necessary tosynergistically or significantly reduce or eliminate conditions orsymptoms associated with a particular disease.

The DEspR -specific antagonist and the one or more other therapeuticagents can be administered simultaneously or sequentially in an amountand for a time sufficient to reduce or eliminate the occurrence orrecurrence of a tumor, a dormant tumor, or a micrometastases. TheDEspR-specific antagonist and the one or more other therapeutic agentscan be administered as maintenance therapy to prevent or reduce thelikelihood of recurrence of the tumor.

As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents or other anti-cancer agentswill be generally around those already employed in clinical therapies,e.g., where the chemotherapeutics are administered alone or incombination with other chemotherapeutics. Variation in dosage willlikely occur depending on the condition being treated. The physicianadministering treatment will be able to determine the appropriate dosefor the individual subject.

In addition to the above therapeutic regimes, the subject can besubjected to radiation therapy.

In certain embodiments of any of the methods, uses and compositionsdescribed herein, the administered DEspR antibody is an intact, nakedantibody. However, in some embodiments, the DEspR antibody can beconjugated with a cytotoxic agent. In certain embodiments of any of themethods and uses, the conjugated DEspR antibody and/or DEspR antibodyfragment thereof is/are internalized by the cell, resulting in increasedtherapeutic efficacy of the conjugate in killing the cancer cell towhich it binds. In some embodiments, the cytotoxic agent conjugated tothe DEspR antibody and/or DEspR antibody fragment thereof targets orinterferes with nucleic acid in the cancer cell. Examples of suchcytotoxic agents include maytansinoids, calicheamicins, ribonucleasesand DNA endonucleases, and are further described elsewhere herein.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present application shall have the meanings that arecommonly understood by those of ordinary skill in the art to which thisdisclosure belongs. It should be understood that this invention is notlimited to the particular methodology, protocols, and reagents, etc.,described herein and as such can vary. The terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims. Definitions of common terms in immunology, andmolecular biology can be found in The Merck Manual of Diagnosis andTherapy, 18th Edition, published by Merck Research Laboratories, 2006(ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The Encyclopediaof Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology andBiotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by WernerLuttmann, published by Elsevier, 2006. Definitions of common terms inmolecular biology are found in Benjamin Lewin, Genes IX, published byJones & Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew etal. (eds.), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers(ed.), Maniatis et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982);Sambrook et al., Molecular Cloning: A Laboratory Manual (2 ed.), ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989);Davis et al., Basic Methods in Molecular Biology, Elsevier SciencePublishing, Inc., New York, USA (1986); or Methods in Enzymology: Guideto Molecular Cloning Techniques Vol.152, S. L. Berger and A. R. KimmerlEds., Academic Press Inc., San Diego, USA (1987); Current Protocols inMolecular Biology (CPMB) (Fred M. Ausubel, et al. ed., John Wiley andSons, Inc.), Current Protocols in Protein Science (CPPS) (John E.Coligan, et. al., ed., John Wiley and Sons, Inc.) and Current Protocolsin Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and Sons,Inc.), which are all incorporated by reference herein in theirentireties.

As used herein, the term “comprising” means that other elements can alsobe present in addition to the defined elements presented. The use of“comprising” indicates inclusion rather than limitation.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular. As usedin this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural references unless the context clearlydictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages canmean ±1%.

It should be understood that this invention is not limited to theparticular methodology, protocols, and reagents, etc., described hereinand as such can vary. The terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the present invention, which is defined solely by the claims.

All patents and other publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that could beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments is based on the information available to the applicants anddoes not constitute any admission as to the correctness of the dates orcontents of these documents.

This invention is further illustrated by the following examples whichshould not be construed as limiting.

EXAMPLES Example 1

Development of Novel Anti-Human Dual Endothelin-1/VEGFsp Receptor(anti-hDEspR) Monoclonal Antibody Treatments as Inhibitors of TumorAngiogenesis and Tumor Cell Invasiveness

DEspR is a key angiogenesis player in embryonic development as seen inDEspR^(−/−) knockout mice (Herrera et al. 2005), and contributes toadult tissue vascularity as seen in adult haplo-deficient (+/−) miceexhibiting decreased tissue vascularity shown by power Doppler analysis(FIG. 2).

Based on the association of tumor invasion and metastasis with intrinsicand evasive resistance to VEGF-targeted therapies, the combination ofanti-invasive and anti-metastatic drugs with anti-angiogenesis therapiesis important to analyze (Bergers and Hanahan 2008). This new therapeuticmandate for anti-cancer therapies can be addressed through a noveltherapy comprising DEspR-inhibition, since DEspR and VEGFsp expressionare detected in human endothelial cells, increased in tumor vessels,detected in cancer cells in tumor tissue arrays and in differentestablished metastatic cancer cell lines, and since inhibition of DEspRdecreases both angiogenesis and tumor cell invasiveness usingcorresponding established in vitro assays, as shown herein.

DEspR and VEGFsp were detected by immunostaining in umbilical veinendothelial cells (HUVECs) and microvascular endothelial cells (HMECS)in both basal and angiogenic tube-formation conditions (FIGS. 3A-3E).Importantly, inhibition of angiogenesis neovessel tube length was seenusing both anti-DEspR (Abl) and anti-VEGFsp (Ab2) antibodies in HUVECs(FIG. 3D) and HMECs (FIG. 3E) angiogenesis assays (Tukey's pairwisemultiple comparison P<0.001 for both HUVECs and HMECs). Similar findingswere observed using other angiogenesis parameters, such as neovesselbranching and inter-connections made. Equally important, DEspR andVEGFsp were also detected in tumor cells, with colocalization of VEGFspand DEspR in the cell membrane and nuclear membrane. Representativeimmunostaining is shown in FIGS. 3A-3C.

DEspR cell-membrane and nuclear-membrane expression were detected inmultiple tumor cell types, indicating that anti-DEspR therapy iseffective for different cancer types. Briefly, DEspR expression wasdetected in human lung non-small cell ca NCI-H727, lung giant cell tumorTIB-223/GCT; breast adenoca MDA-MB-231 (FIGS. 4A-4C) & MDA-MB-468,bladder ca 253J BV, colon adenoca SW480, hepatocellular ca HEP3B,melanoma SK-MEL-2, osteosarcoma MG-63, ovarian adenoca HTB-161/NIH:OVCAR3, prostate adeno ca PC-3mm2, and pancreatic ca CRL-1469/PANC-1 (FIGS.4D). DEspR expression was not detected in HCI-H292 lung mucoepidermoidca, and HEPG2 hepatocellular ca (FIG. 5A), and CCL-86/Raji Burkitt'slymphoma, thus showing specificity of positive observations. Findings inNCI-727 lung ca cells (FIGS. 5B) were corroborated on tumor-sectionimmunostaining of Gr.III lung adenoca (FIGS. 5C).

As shown in FIGS. 6A-6B, in contrast to control (C) and pre-immune abtreatment (PI), DEspR-inhibition via anti-humanDEspR antibody treatmentinhibits tumor cell invasiveness in two cell lines tested, metastaticbreast tumor MDA-MB-231 and pancreatic adenocarcinoma PANC-1 cell lines.The ability to target both tumor angiogenesis and tumor cellinvasiveness through DEspR inhibition can more effectively addresscombined angiogenesis-metastasis phenotypes seen in aggressive tumorsand in evasive resistance to current anti-VEGF therapies.

In vivo proof has also been demonstrated in an irradiation-inducedmammary tumor model in immunocompetent rats using anti-ratDEspR antibody(Herrera et al. 2005). As shown in FIG. 7, anti-DEspR treated ratsexhibited minimal tumor growth compared with mock-treated controls.

Concordantly, immunohistochemical analysis of mammary tumors showedDEspR expression in tumor cells (FIG. 8A) similar to human MDA-MB231breast cancer cells, with no expression in normal breast tissue (FIG.8B). Importantly, residual tumors in treated rats exhibitednormalization of blood vessels (FIG. 8C) in contrast to mock-treatedtumors which showed disrupted endothelium in tumor vessels withencroachment of tumor cells into the lumen (FIG. 8D).

Clinically, the addition of VEGFsp/DEspR-targeted anti-angiogenictherapies to current VEGF/VEGFR2-targeted therapies can additively orsynergistically lead to the desired endpoint of increasing overallsurvival in cancer patients. Given that there are several VEGF/VEGFR2therapies already in the clinics, the translational development ofanti-DEspR therapy as described herein is done in order to provide thisaddition.

Logistically, the experiments described herein demonstrate successfuldevelopment of precursor polyclonal anti-rat DEspR antibodies (FIGS. 7and 8A-8D; Herrera et al. 2005) and polyclonal anti-human DEspR ab(FIGS. 5A-5C and 6A-6B; Glorioso et al. 2007) that exhibit robustaffinity, specificity and functionality.

There are key advantages for selecting the human monoclonal antibodytherapy approaches described herein for DEspR-targeted anti-angiogenesistherapy and target-specific molecular imaging. Humanized/all humanmonoclonal antibody therapies (Ab-Rx) are a rapidly growing class ofhuman therapeutics (Carter 2006) and have a relatively high success rateat 18-24% compared to new chemical entities, including small-moleculeagents at 5% (Imai & Takaoka 2006).

We have developed and validated a murine monoclonal antibody specificfor human-DEspR, termed herein as the 7C5B2 antibody, using a 9-aminoacid (aa)-long epitope located in the extracellular amino-terminal endof hDEspR (Glorioso et al., 2007).

Briefly, mice were immunized with a KLH-conjugated antigenic peptidecomprising the NH₂-terminal 9 amino acids of hDEspR, i.e., DEspR(1-9).After four injections, sera were collected for screening of antibodytiter using free antigenic peptide as antigen. The mouse exhibiting thebest titer was used for fusion experiments. Supernatants of fused cloneswere screened by ELISA using free antigenic peptide as antigen. Allpositive clones were transferred onto 24-well plate andre-tested/confirmed by ELISA. The 10 best clones were selected forfurther testing, which comprised the candidate monoclonal antibodies,anti-hDEspR monoclonal antibody. Relative affinities of prospectivemonoclonal antibodies were determined by ELISA using the supernatantfrom 10 best clones identified.

Analysis of relative monoclonal antibody affinity for antigenic hDEspR9-aa peptide identified clones 7C5C5 and 7C5B2 as the monoclonalantibodies with strongest affinity. These two were selected forexpansion and subsequent large-scale production based upon their higheraffinity for the antigenic peptide.

To ascertain specificity, low-(5G12E8), mid-(2E4H6), and high-affinity(7C5B2) monoclonal antibodies were tested for western blot analysis bytesting the subclone supernatant, and the subsequent purified antibody.Candidate anti-hDEspR monoclonal antibodies were specific for thepredicted 10 kD protein for hDEspR. Western blot analysis was done usingtotal cellular protein isolated from Cos1 hDEspR-transfected cells asantigen, primary antibody comprised purified antibody and subclonesupernatant of 3 selected clones, 10% gel concentration in order todetect the expected 10 kD molecular weight protein of hDEspR.Nitrocellulose (PIERCE) was used with a transfer buffer of 3.07 g Tris,14.4 g Glycine, 200 ml methanol, 800 ml dH2O. HRP-anti mouse polyvalentimmunoglubulins were used (Sigma #0412) 1:100,000; ECL reagent(SuperSignal West Femto Kit #34094), Stain reagent Kodak RP-X-Omat, andx-film (Kodak X-film #XBT-1).

The Western blot results demonstrated specificity of anti-hDEspRmonoclonal antibody regardless of relative affinity, and identified morethan one successful anti-hDEspR monoclonal antibody. Of the antibodiestested, the monoclonal antibody clone with highest relative affinity andspecificity was clone 7C5B2.

The top candidate anti-hDEspR monoclonal antibodies were tested forinhibition of angiogenesis parameters in order to identify candidateanti-hDEspR mAb-Rxtic as anti-angiogenic using established in vitroassays.

To assess anti-angiogenic properties specific to human cells,commercially available, pre-validated established angiogenesis assaysbased on human umbilical vein cells (HUVECs) were used. Multiple invitro-assay angiogenesis parameters were monitored, such as number ofangiogenic tubes formed, ability of “neovessels” or tubes to branch (#branch points), ability of said neovessel branches to connect and formcomplex connections (# branch=connections), and robustness ofangiogenesis represented by neovessel tube length (tube length in mm)Purified 7C5B2 anti-DESPR monoclonal antibody's ability to inhibitHUVECS angiogenic capacity in vitro was assessed accordingly.

An optimal effective concentration of anti-hDEspR 7C5B2 monoclonalantibody that can inhibit >80% of neovessel tube length and number ofbranch points was first assessed. This optimal inhibitor concentrationfor anti-angiogenesis efficacy was found to be 500 nM of the anti-hDEspR7C5B2 monoclonal antibody. This concentration was then used in a seriesof experiments to evaluate other in vitro parameters of angiogenesis.

The anti-hDEspR 7C5B2 monoclonal antibody effectively inhibiteddifferent in vitro parameters of angiogenesis, such as number ofneovessel tubes formed, branch points, branch connections and tubelength. The anti-hDEspR 7C5B2 monoclonal antibody worked as well if notbetter than a previously validated polyclonal antibody, thus validatingits potential as a monoclonal therapeutic.

The anti-hDEspR 7C5B2 monoclonal antibody was also tested for specificbinding to tumor vessel endothelium and/or tumor cells in human cancertissue arrays. The anti-hDEspR 7C5B2 monoclonal antibody was evaluatedin immunohistochemical analyses of human tumor tissue-arrays comprisedof core biopsy specimens representing tumors and normal tissue on thesame slide. Conditions that optimized specificity and sensitivity ofdetection using formalin-fixed, paraffin embedded core biopsy sectionswere tested. Double-immunofluorescence experiments were performed inorder to evaluate hDEspR expression and CD133 expression, with thelatter serving as a marker for putative cancer stem cells.Antigen-retrieval was performed and used anti-hDEspR monoclonal antibodyat 1:10, and commercially available anti-CD133 mAb at 1:20 dilution.

As shown in FIGS. 14A-14B, representative immunohistochemical analysisof human tumor tissue-arrays using anti-hDEspR 7C5B2 monoclonal antibodydetected increased expression of hDEspR in stage II-lung cancer tumorcells (FIG. 14A). Some tumor cells are double immunostain-positive forboth hDEspR and CD133, with other tumor cells immunostained for CD133.These observations demonstrate that hDEspR is also present in postulatedCD133-positive cancer stem cells, as well as CD133-negative tumor cells.In contrast, normal lung specimen does not exhibit any immunostainingfor hDEspR or CD133 (FIG. 14B). In addition, increased DEspR expressionwas observed in a variety of CD133+ cancer stem cell subsets, asdetected by immunofluorescence with a combination of anti-DEspR,anti-CD133 and anti-CXCR4 monoclonal antibodies, including NBCmda-mb-231 cells, pancreatic ductal adenoca Pancl cells, glioblastomacells, and breast cancer cells. Accordingly, in some embodiments, thecompositions and methods described herein can be used in targetedtreatments for tumor resistance and/or recurrence by targeting cancerstem cells or cancer initiating cells.

Accordingly, to summarize, this murine antibody “7C5B2” exhibited highaffinity binding by ELISA to the 9 aa-long epitope (FIG. 9),demonstrates specificity by western blot (FIG. 10), immunostains HUVECsundergoing tubeformation (FIGS. 3A-3E), and pancreatic adenoca PANC-1,and breast cancer MDA-MB-231 cells.

We demonstrated functional efficacy in vitro by showing that both thepolyclonal (Pab) and monoclonal anti-DEspR 7C5B2, specific for humanDEspR, inhibit different parameters of angiogenesis in HUVECs (FIGS.10A-10C): mean number of branchpoints as a measure of neovesselcomplexity (FIG. 10A), and total length of tubes as a measure ofneovessel density (FIG. 10B). Dose response curve for inhibition (FIG.10C) showed equivalent robustness to inhibit both angiogenesisparameters. Importantly, murine 7C5B2 also inhibits tumor cellinvasiveness in MDA-MB-231 human breast cancer and PANC-1 pancreaticcancer cell lines.

This murine anti-human DEspR monoclonal antibody 7C5B2 is thus shown tohave high affinity, specificity, and functionality serves as thestarting antibody for the development of anti-DEspR compositede-immunized all human antibodies, as described herein.

Accordingly, described herein are the development, characterization, andin vitro efficacy testing of anti-hDEspR composite de-immunized allhuman monoclonal antibody (cdHMAb) for use as novel antibody therapiesaimed at addressing evasive and intrinsic resistances to currentanti-VEGF/VEGFR2 antiangiogenic therapies.

We have selected Antitope's Composite Human Antibody technology togenerate anti-hDEspR deimmunized human monoclonal antibodies forantibody therapeutics (Antitope, 2010). This technology generatesde-immunized 100% human antibodies at the outset, in contrast tonon-deimmunized human antibodies derived from phage and transgenic micetechnologies. Briefly, composite human antibodies comprise multiplesequence segments (“composites”) derived from V-regions of unrelatedhuman antibodies are selected to maintain monoclonal antibody sequencescritical for antigen binding of the starting murine precursor anti-humanDEspR monoclonal antibody, and are filtered for the presence ofpotential T-cell epitopes using proprietary “in silico tools” (Holgate &Baker 2009). The close fit of human sequence segments with all sectionsof the starting antibody V regions and the elimination of CD4+ T cellepitopes from the outset circumvent immunogenicity in the development of‘100% human’ therapeutic antibodies while maintaining optimal affinityand specificity through the prior analysis of sequences necessary forantigen-specificity (Holgate & Baker 2009) Immunogenicity can hinderclinical applications of 100% human monoclonal antibodies (Chester etal. 2009).

Briefly, “composite human antibodies” comprise multiple sequencesegments (“composites”) derived from V-regions of unrelated humanantibodies that are selected to maintain monoclonal antibody sequencescritical for antigen binding of the starting murine precursor anti-humanDEspR monoclonal antibody, such as 7C5B2 antibody, and which have allbeen filtered for the presence of potential T-cell epitopes using “insilico tools” (Holgate & Baker, 2009). The close fit of human sequencesegments with all sections of the starting antibody V regions and theelimination of CD4+ T cell epitopes from the outset allow thistechnology to circumvent immunogenicity in the development of ‘100%human’ therapeutic antibodies while maintaining optimal affinity andspecificity through the prior analysis of sequences necessary forantigen-specificity (Holgate & Baker 2009).

As described herein, structural models of mouse anti-hDEspR antibody Vregions were produced using Swiss PDB and analysed in order to identifyimportant “constraining” amino acids in the V regions that were likelyto be essential for the binding properties of the antibody. Residuescontained within the CDRs (using Kabat definition) together with anumber of framework residues were considered to be important. Both theV_(H) and V_(L) (V_(K)) sequences of anti-hDEspR, as described herein asSEQ ID NO: 4 and SEQ ID NO: 9, comprise typical framework residues andthe CDR1, CDR2, and CDR3 motifs are comparable to many murineantibodies.

From the above analysis, it was determined that composite humansequences of anti-hDEspR can be created with a wide latitude ofalternatives outside of CDRs but with only a narrow menu of possiblealternative residues within the CDR sequences. Analysis indicated thatcorresponding sequence segments from several human antibodies could becombined to create CDRs similar or identical to those in the murinesequences. For regions outside of and flanking the CDRs, a wideselection of human sequence segments were identified as possiblecomponents of novel anti-DEspR composite human antibody V regions foruse with the compositions and methods described herein (see, forexample, Table 1).

Based upon these analyses, a large preliminary set of sequence segmentsthat could be used to create novel anti-DEspR composite human antibodyvariants were selected and analysed using iTope™ technology for insilico analysis of peptide binding to human MHC class II alleles (Perryet al 2008), and using the TCED™ (T Cell Epitope Database) of knownantibody sequence-related T cell epitopes (Bryson et al 2010). Sequencesegments that were identified as significant non-human germline bindersto human MHC class II or that scored significant hits against the TCED™were discarded. This resulted in a reduced set of segments, andcombinations of these were again analysed, as above, to ensure that thejunctions between segments did not contain potential T cell epitopes.Selected segments were then combined to produce heavy and light chain Vregion sequences for synthesis. Exemplary heavy chain V region sequencesprovided herein and generated using the above-described methods includeSEQ ID NO: 13-SEQ ID NO: 17. Exemplary heavy chain V region sequencesprovided herein and generated using the above-described methods includeSEQ ID NO: 18-SEQ ID NO: 19.

In vitro efficacy of the antibodies described herein are assessed byexamining dose response-inhibition of angiogenesis of HUVECs (humanumbilical vein cells) and HMECs (adult human microvascular endothelialcells) in angiogenesis assays (see FIGS. 3A-3E, 10A-10C), which in someembodiments are set-up with co-cultured cancer cells, such as PANC-1 andMDA-MB-231, and in some embodiments in normoxia and hypoxia (2% O2)conditions. Both HUVECs and HMECs are used for the following reasons:HUVECs is the standard in the field, but as these cells are umbilicalvein derived, and adult microvascular endothelial cells (HMECs) are alsoused. In addition, angiogenesis is assessed with co-cultured cancercells, in addition to the fetal bovine serum that is usually added inangiogenesis assays, in order to better simulate angiogenic factors thatcancer cells produce which contribute to evasive and intrinsicresistance.

In some embodiments, since hypoxia is one of the triggers forangiogenesis, and one of the contributing factors suspected ofunderlying evasive resistance to current anti-VEGF therapies, in vitroefficacy assays are conducted in normoxia and in 2% O2 hypoxia.Composite deimmunized monoclonal antibody -mediated inhibition of tumorcell invasiveness in vitro is analyzed using MDA-MB-231 and PANC-1 cellsand by using established quantitative assays. These are also done innormoxia and 2% O2-hypoxia conditions, to test a more aggressive tumorcell phenotype known to be associated with hypoxia.

The effects of anti-hDEspR inhibition are compared to controls, whichcan include untreated controls, isotype controls, murine precursoranti-hDEspR monoclonal antibody controls, and bevacizumab controls. Eachpoint for angiogenesis and tumor cell invasiveness assays are done usingat least 5 replicates. Furthermore, for the top 2 candidate-leads, doseresponse curve inhibition responses are also performed, where eachdosage is studied using at least 5 replicates.

Assays can be analyzed by one way ANOVA and multiple pairwise comparisonto assess significant changes. Mean levels of %-inhibition from controlby each candidate lead (e.g., 5-10) are used to rank them according todifferent assays, and the highest ranked two identifies the top-2 leadscorresponding to best inhibitor of angiogenesis and tumor cellinvasiveness in both for example, normoxia and hypoxia conditions, andin both, for example, MDA-MB-231 and PANC-1 cancer cell linesrespectively.

Tumor array analysis is done to corroborate specificity and sensitivityof each to detect tumor cells and tumor neovessels in tissue arrays ofhuman biopsy core samples form different cancer tissue types. This isperformed on a tissue array panel representing solid tumors from brain,pancreas, lung, breast, ovarian, prostate, bladder, colon, stomach.Results are analyzed for specificity given the same immunochemistryconditions used in validation of the murine precursor anti-hDEspRMab-H1. As shown, there is minimal DEspR expression in normal humanpancreas, whereas in stage IV pancreatic cancer exhibits increased DEspRexpression in pancreatic tumor cells and tumor blood vessels. Thecomposite deimmunized monoclonal antibody candidate leads are ranked andthe top-2 that have the best detection of tumor cells and tumorneovessels with optimal signal to noise ratio in tumor tissue arrayimmunohistochemistry are determined This can be compared to tumor-arrayimmunostaining observations obtained with the murine precursoranti-hDEspR Mab.

In addition to de-immunizing the antibodies described herein using insilico screening of T-cell epitopes to minimize and reduceimmunogenicity, the composite humanized anti-hDEspR compositedeimmunized monoclonal antibodies are tested in vitro for immunogenicityin order to select for the least immunogenic composite all human Mab.Immunogenicity screening can be performed using a representative of 50donors, which has proven to correlate with clinical observations (Baker& Jones 2007).

Immunogenicity testing, along with the other in vitro assays ofspecificity and efficacy allows for the selection of a top anti-hDEspRlead, based on a combination of factors, including best affinity(ELISA), specificity (western blot analysis), in vitro efficacy(inhibition of angiogenesis and tumor cell invasiveness) and lowestimmunogenicity. A priori ascertainment of low immunogenicity byelimination of T cell epitopes in the composite antibody humanizationprocess, and low immunogenicity ascertainment by using ex vivo T cellassay technology are important translational research steps, since highimmunogenicity limits ab therapeutic efficacy (Iwai & Takaoka 2006)despite target-specificity and total humanization as has been discussedin clinical studies for Infliximab, Alemtuzumab (review by Baker & Jones2007).

The top composite deimmunized monoclonal antibodies leads are tested forin vivo efficacy by testing anti-DEspR-mediated inhibition of tumorgrowth, angiogenesis and metastasis in established human cancer cellline xenograft and metastasis models in immuno-compromised mice. Cancertissue types representative of evasive resistance (breast cancer) andintrinsic resistance (pancreatic cancer) as observed in publishedreports are also tested. For example, MDA-MB-231 breast cancer andPANC-1 pancreatic carcinoma cell lines are used, since both can be usedto generate xenograft and metastasis spleen-infusion models. ForMDA-MB-231 orthotopic and metastasis models nude mice are used (Oh etal. 2009, Roland et al. 2009). For PANC-1 xenograft subcutaneous modelsnude mice are used as described (Zheng et al. 2008) and NOG mice forPANC-1 metastasis model as described (Suemizu et al. 2007).

Through the strategic use of anti-humanDEspR-specific (e.g., compositedeimmunized monoclonal antibody primary lead) andanti-human-VEGF-specific (bevacizumab) antibodies, and amurine-DEspR-specific Mab, 1) efficacy of anti-DEspR therapy comparedwith anti-VEGF therapy alone can be assessed, and 2) determination ofsynergistic efficacy using a combination of anti-DEspR and anti-VEGFantibodies.

Treatment in xenograft models begin when tumors are 200-300 mm in sizeto simulate clinical cancer therapy scenarios. To assess anti-DEspRtherapy efficacy in metastasis models, a sustained treatment regimenbegun 5 days after the intrasplenic infusion of cancer cells isassessed, as described (Oh et al. 2009). To assess whether anti-DEspRtherapy induces increased risk for metastasis observed with sunitinib(Ebos et al. 2009), Ebos's experiment are performed, wherebyanti-murineDEspR Mab is infused daily for 7 doses beginning 7 days priorto cancer cell infusion. 250 ug is used for each antibody-therapeuticgiven IP 2×/week as described for bevacizumab (Roland et al. 2009), and3×per week for anti-DEspR (Herrera et al. 2005).

Treatment outcomes are assessed by multifaceted parameters: serialimaging of tumor volume and tumor angiogenesis for orthotopic mammaryand subcutaneous pancreatic tumors by, for example, high-resolutionVevo770 ultrasound imaging and power Doppler analysis. Overall survivalis determined, and at this endpoint, repeat ultrasound imaging andhistological analysis of tumor size and angiogenesis is done, along withhistological analysis of malignancy phenotype: nuclear grade, tumor cellinvasion of stroma, tumor cell vascular mimicry, loss of integrity oftumor neovessels and macrophage infiltrates.

Heterozygous DEspR+/−mice live beyond 1 year and breed, which is incontrast to VEGF+/−haplodeficiency which is embryonic lethal at E11.5.However, since adverse effects have been observed in patients onanti-VEGF (bevacizumab) and anti-VEGFR2 (sunitinib, sorafanib)therapies, the anti-humanDEspR-specific antibodies described herein arealso tested for these effects. Analysis of parameters of potentialadverse effects are done in PANC-1 and MDA-MB-231 xenograft modelstreated with cdHMAb-H1 and mDEspR-Mab. For example, potential a)cardiotoxicity can be monitored by serial non-invasive ultrasoundcardiac function analysis; b) hypertension can be monitored by tail cuffBP; c) bowel perforation can be monitored on post-mortem anatomicalinspection at endpoint; d) bleeding, thrombosis can be monitored byexamination and vascular ultrasound and Doppler flow analysis, and e)toxicity screen can be performed, such as liver function tests, renalfunction tests, complete blood count, blood chemistries at endpoint ofstudy. These parameters are compared in mock-treated age-matched tumormodel controls.

Analysis of Molecular Imaging of Tumor Angiogenesis and Tumor CellVascular Mimicry Changes in Response to Therapy by Contrast-enhancedUltrasound Imaging of DespR-targeted Neovessels Compared withVEGFR2-Targeted Tumor Neovessels

Molecular imaging of angiogenesis in tumors has been demonstrated bycontrast-enhanced ultrasound imaging using anti-VEGFR2 antibody-directedmicrobubbles with imaging and contrastenhanced analysis done using theVisualSonics Vevo770 high-resolution ultrasound system (Willmann et al.2007). We have used this same system to detect anti-DEspRantibody-directed microbubbles in carotid artery disease vasa vasorumangiogenesis in a transgenic rat atherosclerotic model associated withcarotid artery disease progression and stroke risk (Decano et al. 2010).As shown in FIGS. 16A-16D, DEspR-targeted molecular imaging (16A)detects DEspR+ endothelial lesions (16B) and vasa asorum angiogenesis(16C). Quantitation of contrast intensity is done using integratedsoftware (16D).

DEspR-targeted molecular imaging is used to test composite deimmunizedmonoclonal antibodies as the targeting module for molecular imagingapplicable to xenograft tumor cell vascular mimicry, and microbubblesare confined to the vascular lumen. MouseDEspR-specific molecularimaging using composite deimmunized monoclonal antibodies as describedherein is performed in order to monitor mouse-derived tumorangiogenesis, and is compared to VEGFR2-specific molecular imaging. Theobservations described herein provide proof that composite deimmunizedmonoclonal antibodies specific for DEspR can serve as the targetingmodule for molecular imaging of tumor cell vascular mimicry in a mousemodel; that molecular imaging of DEspR expression provides atranslatable diagnostic in vivo imaging modality to assess tumorangiogenesis, and that comparative analysis of DEspR-specific molecularimaging provides new insight into the differential contribution of tumorcell vascular mimicry and tumor angiogenesis.

Both MDA-MB-231 xenograft orthotopic and PANC-1 xenograft heterotopictumor models, as well as a PANC-1 intrasplenic-infusion liver metastasismodel are used for molecular imaging experiments. Isotype-antibodymolecular imaging is used as a control to demonstrate specificity ofDEspR-positive molecular imaging. Identical conditions are followed foranti-DEspR and anti-VEGFR2 molecular imaging in order to validatecomparative analysis. For example, a composite deimmunized monoclonalantibody can be used to target tumor cell vascular mimicry; ananti-DEspR composite deimmunized monoclonal antibody can be used totarget mouse neovessel formation monoclonal antibody in human xenografttumors; anti-VEGFR2 can be used as a comparative benchmark, and anisotype antibody can be used as a negative control.

REFERENCES

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Example 2

Molecular Imaging of Vasa Vasorum Neovascularization via DEspR-targetedContrast-Enhanced Ultrasound Micro-Imaging in Transgenic AtherosclerosisRat Model

Given that carotid vasa vasorum neovascularization is associated withincreased risk for stroke and cardiac events, the in vivo studydescribed herein was designed to investigate molecular imaging ofcarotid artery vasa vasorum neovascularization via target-specificcontrast-enhanced ultrasound (CEU) micro-imaging. Accordingly, molecularimaging was performed in male transgenic rats with carotid arterydisease (CAD) and non-transgenic controls using DEspR (dualendothelinl/VEGFsp receptor)-targeted microbubbles (MB_(D)) and theVevo770 micro-imaging system and CEU-imaging software.

It was found that DEspR-targeted CEU-positive imaging exhibitedsignificantly higher contrast intensity signal (CIS)-levels andpre-/post-destruction CIS-differences in 7/13 transgenic rats, incontrast to significantly lower CIS-levels and differences in controlisotype-targeted microbubble (MB_(S))-CEU imaging (n=8) and in MB_(D)CEU-imaging of 5/5 non-transgenic control rats (P<0.0001). Ex vivoimmunofluorescence analysis demonstrated binding of MB_(D) toDEspR-positive endothelial cells, and association of DEspR-targetedincreased contrast intensity signals with DEspR expression in vasavasorum neovessel and intimal lesions. In vitro analysis demonstrateddose-dependent binding of MB_(D) to DEspR-positive human endothelialcells with increasing %cells bound and number of MB_(D) per cell, incontrast to MB_(S) or non-labeled microbubbles (P<0.0001).

The dual endothelin-1 (ET1)/vascular endothelial growth factor-signalpeptide (VEGFsp) receptor or DEspR (formerly dear gene as deposited inGenBank) [1] plays a key role in developmental angiogenesis deduced fromthe embryonic lethal phenotype exhibited by despr^(−/−) knockout micedue to absent embryonic and extraembryonic angiogenesis, aborted dorsalaorta vasculogenesis, and abnormal cardiac development [2]. Whileexhibiting similar abnormal vasculogenesis and angiogenesis phenotypeswith VEGF^(+/−) haploinsufficient mice, despr^(−/−) null mice exhibitdistinct neural tube phenotypes [2-4]. Consistent with its role indevelopmental angiogenesis, DEspR inhibition results in decreased tumorangiogenesis and tumor growth in adult rat mammary tumors and mousemelanomas [2].

Development of target-specific contrast enhanced ultrasonography(CEU)-imaging, herein referred to as “molecular imaging” of vasculardisease neovascularization is important since carotid artery vasavasorum neovascularization is associated with increased risk for stroke[5,6]. However, successful molecular imaging of vasa vasorum neovesselshas not been reported, although detection by non-targeted CEU-imaginghas [7]. On the other hand, successful molecular imaging in differentdisease models detecting different targets [8,9] has shown the potentialof molecular imaging in different disease contexts, such as αvβ3 intumor and hind limb ischemia angiogenesis [10,11], VEGFR2 in tumorangiogenesis [12], ICAM-1 in transplant rejection [13], L-selectin inmalignant lymphnodes [14], and ICAM-1 and VCAM-1 in atherosclerosis[15], P-selectin in myocardial ischemia [16,17], GIIb/IIIa andfibrinogen in thrombosis [18,19]. Molecular imaging of vascular diseaseneovascularization in studies targeting VEGFR2-, ICAM-1 and VCAM-1 didnot detect vasa vasorum neovessels in a hyperlipidemic rabbit model ofinjury-induced vascular neovascularization[9,20].

Demonstrated herein is molecular imaging of DEspR in carotid arterylesions and expanded vasa vasorum neovessels intransgenic-hyperlipidemic, hypertensive carotid artery disease ratmodel.

Materials and Methods

Animals. In order to facilitate molecular imaging studies ofpathological angiogenesis in vascular lesions or in expanded vasavasorum neovessels, a carotid artery disease rat model withhypertension-atherosclerosis as risk factors, the Tg25[hCETP] Dahl-S ratmodel, Tg25, transgenic for human cholesteryl ester transfer proteinwhich develops accelerated stroke [21] or later-onset coronary heartdisease, was selected [22]. 4-month old transgenic male rats (n=13)projected to be around early-midpoint along the disease course of stroke[21] or coronary atherosclerosis phenotype [22], were studied forDEspR-targeted molecular imaging (n=13). MB_(D)-infused non-transgenic,non-atherosclerotic littermates were studied as negative biologicalcontrols (n=5). Isotype-specific MB_(C)-infused transgenic rats (n=8),with the following subgroups: 4 transgenic rats which exhibitedMB_(D)-specific CEU-positive imaging, and 4 de novo transgenic rats,were studied concurrently as negative imaging controls.

Target-specific CEU-molecular imaging. The Vevo770 high resolutionultrasound system with contrast mode software, and streptavidin-coated“target ready” MicroMarker microbubbles (VisualSonics Inc, Canada)previously validated for molecular imaging of VEGFR2 on tumorangiogenesis in mice was used [12]. To target the microbubble to ratDEspR-positive endothelial cells, target ready-MicroMarker microbubbleswere linked to biotinylated anti-DEspR antibody (MB_(D)) viastreptavidin-biotin coupling. For control, target ready-MicroMarkermicrobubbles were linked to biotinylated, isotype-antibody (MB_(C)).Each bolus comprised of 3-4×10⁸ microbubbles in 200-microliters saline,infused into the rat tail vein over 8-seconds.

CEU-imaging of rat carotid arteries comprised a sequence of steps aimedat optimizing MB-target binding, eliminating confounders, andascertaining reproducible CEU-imaging. Baseline images of the carotidartery were first obtained and immobilized the scanhead to maintain theoptimal B-mode view of the common, external, and internal carotidarteries in one 2D image. One minute after MB bolus infusion, the MBblood pool was documented by B-mode imaging for all rats to ascertain MBinfusion and to demonstrate absence of contrast intensity in surroundingtissue. A wait of 4-5 minutes was taken to allow MB_(D) adherence toDEspR-positive endothelial targets [12], and to allow clearance ofunbound circulating microbubbles [23]. Clearance of most circulating MBsfacilitates detection of increased contrast intensity signals due toadherent MBs validated for detection using the Vevo770 imaging system[23]. Adherent MBs were defined by the loss of contrast-intensity uponacoustic destruction performed using pre-set Contrast Enhanced software(VisualSonics, Inc, Canada) as described [12].

Four regions of interest (ROI) on the carotid artery were monitores: thecommon carotid artery, bifurcation, external and internal carotidarteries. Quantitation of contrast intensity signals (CIS) resultingfrom backscatter of adherent targeted-microbubbles was done usingcontrast-enhanced analysis program validated for the Vevo770 imagingplatform (VisualSonics Inc, Canada) detecting pre- and post-acousticdisruption contrast intensity signals. The contra-lateral carotid arterywas checked immediately, and the same CEU-imaging protocol followed.After a 20-minute interval to allow complete clearance of any residualMBs, a pre-set destruction sequence was performed for subsequentCEU-imaging with isotype-specific MB_(C)s following identicalprocedures. For quantitative comparative analyses, the difference incontrast intensity signals between pre- and post-acoustic destruction,CIS-difference, as well as their respective pre-destruction CIS-peaklevels were studied for each carotid artery per rat.

Histology and Immunofluorescence Staining of Rat Carotid Arteries. AfterCEU-imaging, carotid arteries were collected en bloc preserving thesurrounding tissue around the common (CCA), external (ECA) and internal(ICA) carotid arteries including the carotid artery bifurcation. The ECAwas cut longer than the ICA to be able to distinguish the two.Longitudinal serial sections were obtained per carotid artery (50-100sections) and staining every 10th slide with Masson-trichrome allowedproper orientation and site-specific analyses corresponding to ROIs inCEU-imaging. The flanking serial sections to MT-stained slides ofinterest were then immunostained. Double immunofluorescence staining wasdone on deparaffinized sections via sequential antigen retrieval,treatment to reduce background, blocking, incubation with primaryantibody at 4° C. overnight, secondary antibody incubation overnight at4° C. with AlexaFluor 568 goat anti-mouse IgG and AlexaFluor 488 goatanti-rabbit IgG, washing, and mounting using Prolong Gold with DAPI(Invitrogen, CA). Negative controls were run using rabbit-isotypeantibody for anti-rat DEspR antibody. A Zeiss Axioskop2plus microscopewas used for fluorescence imaging and differential interference contrast(DIC) photomicroscopy to provide morphological information overlay toimmunostained sections. Low 2.5× magnification was used for properorientation and site-specific identification along the carotid artery.

In vitro analysis of MB_(D) and DEspR positiveendothelial cellinteractions Human-specific DEspR-targeted MB_(D)s were made followingidentical procedures for rat-specific DEspR molecular imaging with theexception of the use of an anti-humanDEspR monoclonal antibody. Fixednumbers of human umbilical vein endothelial cells (HUVECs) were seededonto IBIDI perfusion 6-lane μ-slide VI (ibidiGmbH, Germany). After 24hours, MB_(D)-type microbubbles were infused at the following MB-cellratios: 8×, 80×, and 800×. Negative controls comprised of 800× MB_(C)sand 800× non-targeted microbubbles, MB_(O)s. These were all infused at20 dynes/cm² shear stress 1-way flow on the same 6-lane micro-flowchamber slide. After 45 minutes of incubation, DAPI nuclear staining wasperformed and excess MBs were washed with HUVECs media at same shearstress. Phase contrast and epifluorescence microscopy was performed in 6random high power fields. Cells and microbubbles were documented byphotomicroscopy and counted as to per cent cells with bound MB, andnumber of MBs per cell. We compared MB_(D), MB_(C) and non-targetedmicrobubbles MB_(O).

Statistical analysis. Values are expressed as mean±S.E.M. Data wereanalyzed with Prism 5 statistics software (GraphPad Software Inc, CA).Where applicable, nonparametric ANOVA and Dunn's multiple comparisontests or ANOVA and Tukey's multiple pairwise comparison tests were used.For two group comparison, nonparametric Kruskal Wallis test wasperformed using Prism5 (GraphPad Software Inc, CA).

Results

DEspR-targeted Molecular Imaging of Carotid Artery. Given the need fordetecting vascular disease-associated angiogenesis in carotid arterydisease [5,6], DEspR was tested to determine whether it can serve as anendothelial target for contrast enhanced ultrasonographic (CEU)-imagingof pathological angiogenesis in carotid artery disease lesions or vasavasorum neovascularization. The Tg25 rat model of carotid artery diseasewas used, comparing 4-month old male Tg25 rats projected to be atmidpoint of atherosclerotic disease course [21, 22], with age-matchednon-transgenic male littermates. Compared to coronary artery disease,investigation of carotid artery disease provides a tactical experimentalsystem with less movement artifacts.

Using the Vevo770 ultrasound contrast-enhanced imaging system andDEspR-targeted microbubbles (MB_(D)) compared with controlisotype-microbubbles (MB_(C)), MB_(D)-specific CEU-positive imaging wasdetected in different regions-of-interest (ROI) along the common carotidartery (CCA), carotid artery bifurcation, proximal internal and/orexternal carotid arteries in 7/13 transgenic rats. MB_(D)-specificCEU-positive imaging was defined as stably increased contrast intensitysignals detected after circulating microbubbles have cleared, and whichdecreased upon acoustic destruction (FIG. 19A). The peak pre-destructioncontrast intensity signals and the differences in pre-/post-destructioncontrast intensity signals (CIS-differences) were significantly higherin MB_(D)-specific CEU-positive images (FIG. 19A, Table 2) compared withCEU-imaging observed in isotype MB_(C)-infused rats (FIG. 19B) and inMB_(D)-infused non-transgenic control rats (n=5), with the latter twoempirically defining CEU-negative imaging. Notably, of the 7 transgenicrats exhibiting MB_(D)-specific CEU-positive imaging, four exhibitedCEU-positive imaging in both carotid arteries, while three exhibitedCEU-negative imaging on the contra-lateral carotid artery, suggestingselectivity of MB_(D)-specific CEU-positive imaging and concordant withspecificity (Table 2). Moreover, six transgenic rats exhibitedCEU-negative imaging with low peak contrast intensity signals,“flat-line” pre-/post-destruction CIS-plot pattern, and minimalCIS-differences (FIGS. 19D, 19E, Table 2) similar to CEU-negativeimaging observed in MB_(C)-control rats (FIG. 19B) and in MB_(D)-infusednon-transgenic controls (FIG. 19C).

Altogether, these observations provide compelling evidence thatMB_(D)-based CEU-positive images are specific and due to adherentMB_(D)s in said carotid arteries. Statistical analysis by one wayanalysis of variance (ANOVA) and post-hoc multiple comparison testingestablish that the CIS-differences of MB_(D)-specific CEU-positiveimaging are significantly higher, P<0.0001, compared to eachCEU-negative imaging study group, respectively (Table 2, FIG. 19D).Interestingly, since CEU-positive imaging is detected only in transgenicrats, and with 54% of transgenic rats exhibiting MB_(D)-specificCEU-positive imaging at 4 months of age equivalent to an early-midpointof the typical model disease course in males [21, 22], averageCIS-differences are significantly different (P<0.0001) betweentransgenic rats and their non-transgenic controls (FIG. 19E). With 7/13transgenic rats exhibiting CEU-positive imaging, and 6/13 exhibitingCEU-negative imaging upon MB_(D) infusion, a sub-grouping of transgenicrats based on MB_(D) CEU-imaging CIS-differences at the 4-month midpointof the disease course is apparent (FIG. 19E).

Interestingly, the CIS-plots of three transgenic rats with the highestMB_(D)-specific CIS-differences exhibited the expected post-acousticdestruction drop in signal intensity but had secondary peaks of contrastintensity signals followed subsequently by decline to low/baselinelevels (FIGS. 20A-20H). This post-acoustic destruction/disruptionpattern is consistent with a particular sequence of microbubble events:microbubble fragmentation accounting for the drop, residual microbubbleacoustic stimulation accounting for the secondary peak, followed byacoustically driven diffusion accounting for the subsequent steadydecline to baseline levels.

Histological analysis detects MB_(D)-microbubbles on DEspR-positiveendothelial cells. Unexpectedly, Masson-trichrome stained histologicalanalysis detected a few microbubbles still attached to endothelial cellsor within intimal lesions (FIG. 21A) obtained from R1:MB_(D) rat withCEU-positive imaging shown. Corresponding DEspR-immunostaining on theadjacent serial section confirmed adherence of MB_(D)-microbubbles toDEspR-positive endothelial cells (FIGS. 21B, 21C). Immunostaining withisotype antibody confirms specificity of DEspR-positive immunostaining(FIG. 21D). Altogether, these observations corroborate MB_(D)-bindingand specificity of MB_(D)-binding to DEspR-positive endothelium.Survival of PEG-coated Target-ready MicroMarker microbubbles(VisualSonics, Inc., Canada) through PBS-buffered 4% paraformaldehydefixation, paraffin embedding and deparaffinization parallels ourobservation that PEG-based biomaterials survive fixation, paraffinembedding, deparaffinization and Masson trichrome staining [24].

Histological analysis of R3:MB_(D) rat shown in FIGS. 20A-20H alsodetected increased endothelial DEspR-positive expression and luminalendothelial pathology, as well as marked carotid vasa vasoral expansionby neovascularization (FIG. 21E, 21F) with DEspR-positive expression invasa vasorum neovessel (FIG. 21G). Double-immunofluorescenceimmunostaining with DEspR and α-smooth muscle actin (αSMA) detected someco-localization of DEspR+αSMA-positive immunostaining in carotid arteryvasa vasorum (FIG. 21H).

Increased DESPR-expression is associated with DEspR positivemolecularimaging. To determine whether increased level and/or area ofDEspR-expression is associated with MB_(D)-specific CEU-positive imagingdefined by higher CIS-differences (FIG. 19D) and higher pre-destructionCIS-peak levels (FIG. 22A), double immunofluorescence-staining wasperformed with anti-DEspR and anti-α-smooth muscle alpha actin (αSMA)antibodies, the latter serving as a positive control for immunostainingof vascular smooth muscle cells in the media. Serial sections fromrepresentative rats were analyzed (n=3/group) with MB_(D)-specificbilateral CEU-positive imaging, MB_(D)-infused bilateral CEU-negativeimaging, and with one-sided CEU-positive/CEU-negative imaging. Analysisof immunofluorescence and differential-interference contrast(DIC)-microscopy showed that MB_(D)-specific CEU-positive imaging isassociated with DEspR+ expression in carotid intimal lesions, vasavasorum neovascularization and DEspR+ expression in vasa vasorumneovessels (FIGS. 21B, 21C, 22B, 22C, Table 2). In contrast, rat carotidarteries exhibiting MB_(D)-CEU-negative molecular imaging wereassociated with minimal, if any, DEspR+ endothelial expression (FIG.22D, Table 2). Low levels of αSMA expression in carotid media smoothmuscle cells (SMCs) compared with the expanded vasa vasorum were alsonoted (FIG. 22A), due, without wishing to be bound or limited by atheory, most likely to the synthetic state of SMCs in these hypertensiverats, since αSMA expression is deinduced in synthetic or proliferatingSMCs [25]. These observations link MB_(D)-specific CEU-positive imagingin this rat model with increased DEspR expression intensity and area inboth intimal lesions and vasa vasorum neovessel density.

In vitro analysis of dose-response MB_(D)-adherence to DEspRpositiveendothelial cells. In order to further dissect MB_(D)interactions with DEspR-positive cells, the dose-response of MB_(D)adherence in vitro was tested. In order to avail of standardized primarycultures of endothelial cells and to gain translational insight intomolecular imaging in humans, human umbilical vein endothelial cells(HUVECs) which express DEspR in proliferating and pro-angiogenesisculture conditions as detected by a human-specific anti-DEspR monoclonalantibody were used. Using increasing number of MB_(D)s from 8×, 80×, and800×MB_(D) to cell ratio, it was observed that HUVECs are increasinglybound by MB_(D)s being 100% bound at 80x MB_(D):cell ratio (FIGS.23A-23C), in contrast to 800x MB_(C)s (FIG. 23D) and non-targetedMB_(O)s (FIG. 23E) which bound 6.8% and 8.2% of HUVECs respectively(FIG. 23F). Moreover, analysis of number of MBs bound per cell after a45-minute incubation and wash at flow rates with aortic-like shearstress of >20dyne/cm² revealed significant differences in number of MBsbound per cell increasing from 8x, 80x to 800x as follows: 2.3, 17 and49 MBs/cell, with only 0.6 and 1.1 MB/cell for non-targeted MBs andisotype MB_(C)s (ANOVA P<0.0001). These observations reflect therelative stability and specificity of the MB-cell interaction.Importantly, cell toxicity was not observed upon contact of MB withcells even at high-dose 800x MB_(D).

Although VEGFR2-targeted molecular imaging of tumor angiogenesis hasbeen reported [12], previous VEGFR2-targeted molecular imaging of vasavasorum neovascularization was not successful, along with other vascularadhesion molecule targets, leading authors of these reports to suggestthat vasa vasoral flow might be a technical hurdle for target-specificCEU-molecular imaging [9]. Accordingly, the molecular imaging ofDEspR-positive endothelial cells in carotid artery disease demonstratedherein (FIGS. 19A-22E) provide novel research and diagnostic tools forin vivo molecular imaging of carotid artery disease endothelium andexpanded vasa vasorum. Without wishing to be bound or limited by theory,given optimal ultrasound imaging parameters, the likely factors fordifferential success in target-specific CEU-molecular imaging could bedifferences in molecular thresholds defined by the level and/or area ofexpression of the target, and/or in technical thresholds defined bydensity and size of, as well as flow in target vessel(s). Thesethresholds must be surpassed concurrently for detectable targetedCEU-positive imaging or molecular imaging. More specifically, the levelof DEspR expression, the degree of luminal endothelial pathology, andthe density of vasa vasorum neovascularization, along with the largersize of the rat carotid artery disease model used here, comprise factorscontributing to successful DEspR-targeted CEU-positive imaging ofcarotid artery vasa vasorum in the Tg25 rat model of carotid arterydisease, in contrast to the negative molecular imaging results targetingVEGFR2 reported for vasa vasorum neovascularization in a carotid arteryinjury-induced mouse model [9]. Furthermore, differences betweenCEU-positive transgenic rats from CEU-negative transgenic rats reveal aputative threshold for CIS-differences (FIG. 19E) and pre-disruptionCIS-peak levels (FIG. 22A). This observed threshold for CEU-positiveimaging provides evidence that DEspR-targeted CEU-positive imaging canbe a non-invasive biomarker for pathological angiogenesis, and havepredictive value for disease progression.

Surpassing the molecular and technical threshold for successfuldetection of target-specific molecular imaging is concordant with theprinciple that reflectivity is directly proportional to theconcentration of the microbubbles themselves [26]. More specifically,greater DEspR-expression and greater density of DEspR-positiveendothelial cells, be it at the lumen or in vasa vasorum, can translateto greater concentration of bound microbubbles in the methods describedherein. This in turn, without wishing to be bound or limited by theory,is expected to translate to greater reflectivity and detection levelssince microbubble-cell binding does not dampen microbubble reflectivityin contrast to leukocyte engulfment of microbubble [27]. After clearanceof most circulating microbubbles and prior to acoustic disruption,stable binding of target-specific microbubbles exhibits a relativelystable contrast-intensity level that is significantly greater thannegative or background contrast-intensity (FIG. 20d , ANOVA P<0.0001) .Since high-frequency imaging can induce microbubble fragmentation or gasdiffusion per se, a slight decline could also be observed prior toacoustic disruption, without wishing to be bound or limited by theory.However, upon acoustic disruption a drop in contrast-intensity due tofragmentation is observed to confirm microbubble binding (FIGS.19A-19E). Acoustic fragmentation may not be complete due, withoutwishing to be bound or limited by theory, to microbubble interaction inhigh-density ROIs which could dampen microbubble resonance [28], or frominability of microbubbles within microvessels to reach 10-folddiameter-fluctuation that underlies acoustic fragmentation [29].Furthermore, incomplete fragmentation with gas release and relativelylow flow, as would be expected in vasa vasorum compared to carotidartery lumen, without wishing to be bound or limited by theory, couldaccount for the secondary peak observed in rat-R3 followed by slowdecline back to baseline levels. The secondary peak is likely not due torefill because at this experimental time point there is minimal, if any,circulating microbubbles (FIGS. 19A-19E, 20A-20H). The fact that rat-R3reached higher contrast-intensity levels than rat-R1 suggests greatermicrobubble concentration, which can also dampen acoustic destructiondue to inter-microbubble interactions [28]. Notably, while acousticfragmentation corroborates microbubble binding, the pattern of acousticfragmentation or diffusion can also provide further insight intomicrobubble concentration, as well as binding site vessel-caliber andflow. This provides a novel, alternative molecular imaging paradigm tothat reported for mouse aortic root atherosclerosis [30]. WhileCEU-imaging in the current set-up is successful, in other embodiments,non-linear imaging of adherent microbubbles can be used to providegreater sensitivity and/or improved quantitation as observed forintravascular ultrasound for vasa vasorum flow imaging [31].

The detection of dose-dependent increase in % cells targeted by MB_(D)sand dose-dependent increase in number of MBs per cell (FIGS. 23A-23G),gives insight into the stable interaction, kinetics, specificity andnon-toxicity of DEspR-targeted MB-cell interactions. More importantly,given that in vitro studies were performed using human endothelial cellsand human-specific anti-DEspR monoclonal antibody for targeting, thatMB-cell coupling withstood a high shear stress wash after 45 minutes anddid not elicit cell toxicity on contact, these in vitro observations ofMB_(D)-cell interactions demonstrate DEspR-targeted molecular imaging ofpathological angiogenesis as a useful therapeutic and diagnostic tool.

Altogether, comparative analysis of molecular imaging contrast-intensitylevels, histological confirmation of microbubble-to-endothelium binding,immunostaining confirmation that DEspR-positive molecular imaging isassociated with DEspR-positive endothelial cell expression, andconcordant patterns of bound microbubble behavior after acousticdestruction, demonstrate that target-specific molecular imaging ofcarotid endothelium and vasa vasorum neovascularization in carotidartery disease rat model is feasible using the methods and reagentsdescribed herein that target DEspR. The identification of DEspR as asuccessful target for in vivo molecular imaging of vasa vasorumneovascularization and carotid artery disease lesions can facilitate thelongitudinal study of vasa vasorum neovascularization and endothelialchanges in carotid artery disease progression in animal models. Alongwith the in vitro observations of MB_(D)-HUVECs stable binding, the datademonstrate the use of molecular imaging techniques described herein inthe earlier detection of pathophysiological changes in cardiovasculardisease for estimations of risk for disease progression andcomplications.

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TABLE 2 DEspR-targeted molecular imaging in transgenic rat model ofcarotid artery disease Rat groups: 4 m-old male Tg 25+ Non-transgenicMB_(D) Contrast enhanced CEU(+) CEU (−) CEU (−) image # rats: bothcarotid 4 6 5 arteries # rats: one carotid artery 3* 3* — Contrastintensity signal Δ MB_(D) (n = 18 rats) 89.96 ± 11.0*** 2.2 ± 0.9 2.0 ±0.8 MB_(C) (n = 8 rats)  1.9 ± 0.7 ND ND Histopathology: Intimallesions, plaque (+) +/− (−) Vasa vasorum expansion (+) +/− (−)Immunostaining: DEspR (+): in vasa +/− (−) vasorum, initimal lesionsValues are group means ± sem; #, number; Δ, delta or difference; (+),present; (−), absent; +/−, low to no expression; *same 3 rats; ***ANOVAand Tukey's multiple pairwise comparison P < 0.0001. CAD, carotid arterydisease; m, month; MB_(D), DEspR-targeted microbubble; MB_(C),isotype-targeted microbubble.

Example 3

Dual Endothelin-1/VEGFsp Receptor (DEspR) in Cancer: Target for DualAnti-angiogenesis/anti-tumor Cell Invasiveness Therapy

The development of intrinsic and extrinsic resistance to currentanti-VEGF/VEGFR2 therapies have been observed. As described herein,DEspR expression is found to be increased in primary and metastatictumor αSMA-positive and αSMA-negative vascular endothelium, and in tumorcell- and nuclear-membranes of different human cancer tissue types andcell lines. Further, DEspR-inhibition using the human-specificanti-DEspR antibody treatments described herein decreased humanendothelial cell angiogenesis and tumor cell invasiveness. Further, itwas found that ligand-specific DEspR signaling-profiles are distinctfrom VEGF/VEGFR2's. Accordingly, described herein are data demonstratingtargeting of DEsPR for dual tumor-cell and endothelial deliveries, andfor dual anti-angiogenesis/anti-invasiveness therapies.

Introduction

Although the critical role of the angiogenic switch in cancerpathogenesis has been recognized [1], anti-angiogenesis therapiesdirected at vascular endothelial growth factor and/or its receptor,VEGF/VEGFR2-centric anti-angiogenesis therapies, alone or in combinationwith other anti-cancer therapies, have not attained the hoped-fortreatment goal of long-term efficacy such that cancer is reduced to adormant, chronic manageable disease [2-5]. Cumulative observations haveshows that all three FDA-approved VEGF pathway inhibitors (anti-VEGFbevacizumab or Avastin, AntiVEGFR2 sunitinib, and sorafanib) result insignificant but transitory improvements in the form of tumor stasis orshrinkage, and only for certain cancers despite most, if not all cancertypes exhibiting pathological angiogenesis [2,6]. Moreover, whileanti-VEGF pathway therapies have reduced primary tumor growth andmetastasis in preclinical studies [7], recent mouse tumor model studiesreport that sunitinib and an anti-mouseVEGFR2 antibody, DC101, increasedmetastasis of tumor cells despite inhibition of primary tumor growth andincreased overall survival in some cases [8,9]. Cumulative observationsimplicate several mechanisms of intrinsic and evasive resistance, suchas, without wishing to be bound or limited by theories, pre-existingmultiplicity of redundant pro-angiogenic signals; upregulation ofalternative pro-angiogenic pathways, recruitment of bone marrow-derivedpro-angiogenic cells, increased pericyte coverage for the tumorvasculature obviating the need for VEGF signaling, and invasive andmetastatic co-option of normal vessels without requisite angiogenesis[2-5]. Additionally, 10-fold increase in VEGF levels have been detectedupon bevacizumab anti-VEGF therapy in humans [10] and upon anti-VEGFR2ab-therapy in mice [11], which could, without wishing to be bound orlimited by a theory, contribute to evasive resistance.

Both VEGF and VEGFsp (vascular endothelial growth factor signal peptide)originate from the same propeptide, and a 10-fold ‘rebound’ increase inVEGF could, without wishing to be bound or limited by a theory, alsoresult in a concomitant 10-fold increase in VEGFsp, thus resulting in a10-fold increase in VEGFsp's post-cleavage function of activating itsreceptor, the dual endothelin1/VEGFsp receptor or DEspR, formerly calledDear and deposited in GenBank as Dear [12]. DEspR knockout mouseexhibits arrested vasculogenesis and absent angiogenesis resulting inE10.5-E12.5 day embryonic lethality [13]. Concordantly,DEspR-haploinsufficiency resulted in decreased syngeneic melanoma tumorgrowth, and anti-DEspR antibody inhibition decreased tumor growth andtumor angiogenesis in rats with irradiation-induced mammary tumors [13].Furthermore, DEspR's other ligand is endothelin-1 (ET1) [12], and allother known ET1 receptors, ETa and ETb, do not exhibit an embryoniclethal angiogenic phenotype in their respective knockout mouse models[14,15, 16.].

Described herein are novel anti-angiogenic strategies using anti-humanDEspR ab-inhibition and characterizing the murine precursor of ananti-DEspR antibody therapeutic. It was found that DEspR is upregulatedin some solid tumor cells and tumor vascular endothelium, and thathuman-specific anti-DEspR polyclonal and monoclonal antibodies inhibithuman endothelial cell tube formation and tumor cell invasiveness invitro, and that DEspR utilizes ligand-specific signaling pathways knownto mediate angiogenesis and cancer cell invasiveness.

Materials and methods

Cell lines and antibody development MDA-MB-231 and PANC-1 cells wereobtained from American Type Culture Collection (Rockville, Md.).MDA-MB-231 cells were maintained in DMEM media (Sigma Chemical, St.Louis, Mo.) supplemented with 10% FBS, L-glutamine, penicillin, andstreptomycin (GPS). PANC-1 cells were maintained in DMEM (SigmaChemical, St. Louis, Mo.) with high glucose, 10% FBS and GPS. Humanumbilical vein endothelial cells, HUVECs, were obtained from CascadeBiologics, Inc., and maintained in Endothelial Growth Media-2 (EGM-2)containing 2% FBS and GPS. Monoclonal antibody development was customperformed by ProMab Biotechnologies, Inc (Richmond, Calif.) using a nineamino-acid DEspR NH₂-terminal peptide, M₁TMFKGSNE₉ (SEQ ID NO. 20) ofhDEspR as antigen. Screening of hybridoma supernatants and initialcharacterization of candidate monoclonal antibodies were performed byELISA using free hDEspR-antigenic peptide as antigen.

Monoclonal antibody characterization by ELISA and Western blot analysis.The M₁TMFKGSNE₉ (SEQ ID NO: 20) antigenic peptide was coated directly onwells of a microtiter plate. Appropriate dilutions of primary antibodieswere incubated at 37° C. for 1 hr. The wells were then incubated withHRP labeled anti-IgG (SIGMA cat# A0168) at 1:9000 at 37° C. for 1 hr.The reactions were visualized by the addition of3,3′5,5′-tetramethylbenzidine substrate (incubation at 37° C. for 10min) and read spectrophotometrically at 450nm. Western blot analysis wasdone as described [17] using equal amounts of whole cell protein extract(40 μg) from Cos1 cell transfectants stably expressing hDEspR [17] andcorresponding candidate monoclonal antibodies raised against hDEspRspecific synthetic peptide Immunoreactive hDEspR (10 kDa polypeptide)was detected by chemiluminescence using the ECL Western Detection kit(GE Healthcare).

HUVEC tube formation assay for angiogenesis. Validated 2^(nd) passagehuman umbilical vein endothelial cells—HUVECs (Cascade Biologics,Oregon) were obtained and cultured until the 4th passage and were thenharvested at 80% confluence using mild trypsinization. The cell pelletwas then washed twice in serum free media (basal media) containing M-200(Cascade Biologics, Oregon) 1 μg/ml hydrocortisone, 10 ng/ml EGF, 3ng/ml bFGF and 10 μ/ml heparin. Cells were then resuspended in thisserum free media and seeded at 20,000 cells per well (100 μL) onto a 96well plate Angiogenesis System: Endothelial Cell Tube FormationMatrigel™ Matrix (BD Biosciences, MA). Different angiogenic andanti-angiogenic conditions were assayed in quadruplicate as indicatedusing basal media alone or with one or more of the following: 2% FBS, 20nM VEGF, 20 nM VEGFsp, 20 nM ET1. Antibodies used for inhibition wereall affinity purified and used in the following concentrations: 500 nManti-hDEspR polyclonal antibody (Pab), 500nM anti-hDEspR 7C5B2monoclonal antibody (Mab), 500 nM anti-VEGFsp Pab, and for correspondingisotype controls either 500 nM preimmune IgG (75 μg/ml) for Pab, and 500nM IgG2b for anti-hDEspR Mab. Different experimental conditions weretested in quadruplicate as follows: basal media alone (BM), BM with 2%FBS; BM with 20 nM VEGF; BM with 20 nM VEGFsp; BM with 20 nM ET1; BMwith 20 nM VEGF and 500 nM (75m/ml ) pre-immune IgG; BM with 20 nM VEGFand 500 nM anti-VEGFsp; BM with 20 nM VEGF and 500 nM anti-hDEspR; BMwith 20 nM VEGFsp and 500 nM anti-hDEspR; BM with 20 nM ET1 and 500 nManti-hDEspR; BM with 2% FBS and 500 nM anti-VEGFsp; and BM with 2% FBSplus 500 nM anti-hDEspR. In other experiments increasing concentrationsof anti-hDEspR 7C5B2 mAb (0.05-500 nM) were tested. HUVECs were thenincubated in different conditions as specified at 37 ° C. for 16 hours;after which, resulting angiogenic tube formations were viewed under themicroscope and images of ˜70% of the well (central parts) were taken foranalysis. Various parameters were measured for each angiogenic conditionusing ImageJ (NIH—http://rsb.info.nih.gov/ij/) namely total tube length,average tube length, average tube thickness, number of branch pointsdefined as cluster of cells possessing tube-like extensions measuringmore than 2× the length of the cell aggregates, number of connectionsdefined as 3 or more connections between tube-like structures in seriesor parallel and number of closed polygons bounded by the tubularstructures.

Invasion assay. MDA-MB-231 and PANC-1 cell invasion assays wereperformed as described [18] using the BD Bio-Coat Matrigel invasionassay system (BD Biosciences, Franklin Lakes, N.J.). MDA-MB-231 andPANC-1 cells were suspended in growth media and seeded onto pre-coatedtranswell chambers (3×10⁴cells/well). The transwell chambers were thenplaced into 24-well plates, to which basal medium only or basal mediumcontaining various concentration of antibodies were added. Cells wereincubated for 16 hr and the invading cells were fixed and stained withDiff-Quick stain. The number of invading cells per well were countedunder the microscope. Each condition was assessed in four replicates.

Immunostaining of tumor tissue arrays and tumor cells. Human cancer cellline-array DEspR immunostaining was custom-performed by Pantomics, Inc.using our in-house polyclonal human-specific anti-DEspR antibody. Tumortissue arrays were obtained from Pantomics, Inc. and immunostained forDEspR using polyclonal and monoclonal anti-hDEspR antibodies at 1:20after demonstration of concentration-dependent immunostaining 1:10,1:50, 1:100. Deoxyaminobenzidine immunostaining was done using thepolyclonal antibody as described [13]. Double immunofluorescencestaining was done on deparaffinized sections via the following steps:antigen retrieval, treatment to reduce background, blocking, incubationwith primary antibody at 4° C. overnight, secondary antibody incubationovernight at 4° C. with AlexaFluor 568 goat anti-mouse IgG andAlexaFluor 488 goat anti-rabbit IgG, washing, and mounting using ProlongGold with DAPI (Invitrogen). Negative controls were run usingrabbit-isotype antibody for anti-rat DEspR antibody. A ZeissAxioskop2plus microscope was used for fluorescence imaging andphotomicroscopy.

Multiplex analysis of signaling proteins by Ab-microarray. Analysis ofligand-dependent modulation of different signaling pathways by DEspR wascustom performed by Kinexus Corp. (Kinexus, Canada) utilizing the Kinex™Antibody Microarray System spanning 506 phosphoprotein-specificantibodies in duplicates or multiple replicates, as well as 740pan-specific antibodies of signaling molecules. The effects of ET1- andVEGFsp-DEspR activation were analyzed on multiplex signaling pathwaysafter 30 minutes of ligand-treatment (ET1, 10 nM; VEGFsp, 10 nM),compared with the respective non-activated DEspR in non-treatedcontrols, using Cos1-hDEspR permanent cell transfectants. Allfluorescent signals were normalized to background. Data are presented aspercentage change from control (% CFC), or change detected after 30minutes of ET1 or VEGFsp-treatment compared with non-treatedtransfectant-matched controls respectively. The %CFC=[Treated^(Ave)−Control^(Ave)]/Control^(Ave)×100. Although % CFC>25%is suggested as a significant difference, only values exhibiting >50%CFC and with % error range between duplicates less than 20% for bothtest and control samples were presented. The % errorrange=[Duplicate^(n)−Average]/Average. A % error >20% was accepted ifthe % CFC remained >50% using the lesser of the duplicates incalculating % CFC.

Statistical analysis. One way analysis of variance (ANOVA) followed byall pairwise multiple comparison Tukey test were performed afterascertaining normality using SigmaStat 2.03 software package. A P<0.05was considered statistically significant.

Results

DEspR expression is increased in human tumor cells and tumor vessels.DEspR-specific expression patterns were investigated in human cancertissues and cells. Tumor tissue array analysis was performed using ahuman-specific anti-DEspR polyclonal-antibody [17]. Concordant with ratirradiation-induced mammary tumor model observations of rat-specificanti-DEspR antibody [13] immunostaining, immunohistochemical analysis ofDEspR expression in human tumor tissue arrays detected increased DEspRexpression in thin-walled tumor vascular endothelium in hepatic,pancreatic (FIGS. 24A-24F), stomach, breast (FIGS. 25A-25F), colon andlung (FIGS. 26A-26F) cancer, compared with vascular endothelium innormal tissue biopsy cores respectively be it arterial or microvascularendothelium (FIGS. 24A-24F, 25A-25F, and 26A-26F). Notably, vascularendothelium in stomach cancer metastatic foci in the lung (FIG. 25C) andbreast cancer metastatic foci in lymph node (FIG. 25F) also exhibitincreased DEspR immunostaining Moreover, pancreatic (FIGS. 24E, 24F),stomach (FIGS. 25B, 25C), breast (FIG. 25E), lung (FIG. 26C) and colon(FIG. 26E, 26F) tumor cells exhibit increased DEspR expression withsub-cellular localization in the cell membrane, cytoplasm and nuclearmembrane. This increased DEspR expression in tumor neovessels and tumorcells demonstrated herein indicate that that DEspR plays a role in bothtumor neovascularization and in tumorigenesis.

To further confirm expression in tumor cells DEspR-immunostaining ofcancer cell-array testing different types of previously characterized,established cancer cell lines was next performed (Table 3). In contrastto a few cell lines tested with minimal if any DEspR expression, severalcancer cell lines exhibit DEspR expression with nuclear membrane DEspRexpression associated with high-nuclear grade (Table 3, FIGS. 27A-27F).Representative photomicrographs demonstrate tumor cell expression withstrongest DEspR-immunostaining in nuclear membranes of most tumor cells,but not all. The selective nuclear membrane immunostaining (FIGS.27A-27F) confirms specificity of DEspR immunostaining, along withnegative immunostaining of some cancer cell lines (Table 3).Importantly, these observations are concordant with the observations incancer tissue sections described herein (FIGS. 24A-24F, 25A-25F, and26A-26F). Nuclear membrane localization indicates that DEspR can play arole in crosstalk between the cell membrane and nuclear membrane, beyondreceptor-mediated signal transduction.

High-affinity anti-hDEspR monoclonal antibody generated againstN-terminal 9-aa extra-cellular domain. In order to investigateanti-DEspR inhibition as an anti-angiogenic strategy, a human-specificanti-DEspR monoclonal antibody was developed using a 9-aa peptidespanning the N-terminal extracellular domain of human DEspR identical tothe strategy use to develop the human-specific anti-DEspR polyclonalantibody used in DEspR immunostaining (FIGS. 24A-24F, 25A-25F, 26A-26F,and 27A-27F) [17]. From 67 hybridoma clones, a preliminary screenidentified top ten candidate monoclonal antibody hybridoma clones whichwere then analyzed for affinity to the 9-aa peptide N-terminal domain byindirect ELISA (FIG. 28A). Analysis of specificity by Western blotanalysis of mab-mediated binding to hDEspR protein (10 kDa) isolatedfrom Cos1-hDEspR transfectants in contrast to control non-transfectedCos1 cells identified hybridoma clone 7C5B2. As shown in FIG. 28B, 7C5B2anti-hDEspR monoclonal antibody hybridoma clone exhibited specificity asboth “super clone” supernatant and purified monoclonal antibody.Isotyping of 7C5B2 showed that this monoclonal antibody belongs to themurine IgG2b isotype class of antibodies.

Co-localization of DEspR and its ligand, VEGFsp in human umbilicalvascular endothelial cells (HUVECs). Analysis of receptor-ligandco-localization by double immunostaining in HUVECs showed specificdetection of DEspR on endothelial cell membrane cultured inpro-angiogenesis conditions using the anti-hDEspR monoclonal antibody.Double immunostaining detected co-localization of DEspR with its ligandVEGFsp using an anti-VEGFsp polyclonal antibody, thus demonstrating thatanti-hDEspR monoclonal antibody specifically targets DEspR. Anti-DEspRpolyclonal antibody also gave identical results.

Anti-DEspR inhibition by anti-hDEspR polyclonal antibody and 7C5B2monoclonal antibody decrease angiogenesis. The effects of 7C5B2monoclonal antibody inhibition of DEspR on angiogenesis usingestablished in vitro HUVECs-based angiogenesis assays was then assessed.It was first showed that 7C5B2 monoclonal antibody detects cell-membraneDEspR expression in tubes/“neovessels” formed by HUVECs inpro-angiogenesis conditions, thus validating the use of thisangiogenesis assay system. Next, two established parameters of in vitroangiogenesis were analyzed, total tube length and branching ofneovessel-tubes formed by HUVECs in pro-angiogenesis conditions. Usingvarying doses of 7C5B2 monoclonal antibody from 0.05 to 500 nM,concentration dependence of angiogenesis inhibition is demonstrated forboth total tube length and number of branch-points, and identifies 500nM 7C5B2 monoclonal antibody as the full-strength inhibitory dose (FIG.29A). This dose was then applied to repeat independent inhibitionexperiments comparing the newly developed 7C5B2 monoclonal antibody withthe previously characterized anti-hDEspR polyclonal antibody. Comparedto non-treated controls, and pre-immune and IgG2b-isotype-specificnegative controls for polyclonal antibody and 7C5B2 monoclonal antibodyrespectively, 500 nM anti-hDEspR antibody inhibited angiogenesis,measured as total tube length and mean number of branchpoints,significantly (ANOVA with all pairwise multiple comparison Tukey test,P<0.01). Other angiogenesis parameters, number of tubes andbranch-interconnections were also significantly inhibited. Concordantly,a polyclonal anti-VEGFsp antibody also inhibited angiogenesis in HUVECs.

Analysis of anti-hDEspR 7C5B2 monoclonal antibody immunostaining andinhibition of tumor cell invasiveness. Having shown that DEspRinhibition reduces angiogenesis, the efficacy of 7C5B2 monoclonalantibody -mediated anti-DEspR inhibition on tumor cell invasiveness wasnext assessed since DEspR is detected in different tumor cell lines(FIGS. 27A-27F) and cancer tissues (FIGS. 24A-24F, 25A-25F, and26A-26F). Two cancer cell lines representing aggressive breast cancerand pancreatic cancer, MDA-MB-231 and PANC-1 cancer cell linesrespectively, were examined Immunostaining with 7C5B2 monoclonalantibody detected nuclear- and cell-membrane DEspR expression in bothcell lines, as well as cytoplasmic expression. Functional analysisdetected concentration dependent inhibition of tumor cell invasivenessfrom 0.05 to 500 nM 7C5B2 monoclonal antibody, with an EC50 of 3.55±0.32nM. Using 500 nM 7C5B2 monoclonal antibody, DEspR inhibition wasobserved in both MDA-MB-231 (FIG. 30B) and PANC1 (FIG. 30C) cells,compared to control non-treated cells and IgG2b-isotype treated cellsrespectively (ANOVA followed by all pairwise multiple comparison test,P<0.001 and P<0.01 respectively). These observations indicate dualeffects of DEspR inhibition on both angiogenesis (FIG. 29B-29C) andtumor cell invasiveness (FIG. 30B-30C).

Anti-hDEspR 7C5B2 monoclonal antibody-immunostaining of tumor vascularendothelium and tumor cells. Having shown efficacy of DEspR-inhibitionon angiogenesis and tumor cell invasiveness, 7C5B2 monoclonal antibody-immunostaining in breast and pancreatic cancer tissues in contrast tonormal was next evaluated to confirm increased DEspR expression in tumorvascular endothelium and tumor cells as detected using anti-hDEspRpolyclonal antibody (FIGS. 24A-24F, 25A-25F, and 26A-26F), as well as todelineate DEspR-targeting profile of 7C5B2 monoclonal antibody.

Double immunostaining of DEspR and alpha smooth muscle actin (αSMA), totrack microvascular pericytes and cancer tissue stromal myofibroblasts,detected minimal DEspR expression in normal breast tissue blood vesselsand mammary epithelial cells, and normal αSMA expression in mammarymyoepithelial cells and arteriolar smooth muscle cells highlightingminimal to no DEspR expression (FIGS. 31A-31C). In contrast, in arepresentative breast cancer tissue sections of ductal invasivecarcinoma, double immunostaining detected prominent DEspR expression intumor microvascular endothelium, in microvessels and arteriolesco-expressing αSMA, as well as in ductal carcinoma epithelial cells(FIGS. 31D-31F). Increased tumor vascularization is also noted comparedto non-cancer ‘normal’ control tissue (FIGS. 31A-31C).

Similarly, in normal pancreas, minimal DEspR expression is detected inmicrovessels (FIGS. 32A-32C), and in arterial endothelium in contrast tostrong αSMA expression in arterial media smooth muscle cells (FIGS.32C). In contrast, DEspR expression is increased in pancreatic cancerαSMA-negative microvascular and αSMA-positive microvascular andarteriolar endothelium (FIGS. 32D-32E). As observed in breast cancerepithelial cells and in PANC-1 cancer cell line, pancreatic cancerductal carcinoma epithelial cells exhibit marked DEspR-positiveimmunostaining (FIG. 32F).

Phosphoproteome analysis of DEspR signal transduction. Using aphosphoprotein-specific antibody-array, ligand-specific signaltransduction pathways activated by DEspR upon binding to its dualligands, ET1 and VEGFsp respectively in permanent Cos1-cell DEspRtransfectants were identified (Table 4). Cos1 cells were used as thesecells do not have endogenous DEspR, ET1 or VEGFR2 expression.Non-treated and treated Cos1-DEspR transfectants were compared. As shownin Table 4, regardless of ligand, DEspR's phosphoproteome (limited tosignaling phosphoproteins with >50% CFC) activates signaling pathwaysknown to be involved in mechanisms of angiogenesis, tumor cellinvasiveness or metastasis. Additionally, some DEspR-phosphorylatedsignaling molecules for either ET1 or VEGFsp-activation of DEspR havebeen directly linked to either neuronal or hematopoietic stem cells,with some also implicated in cancer stem cell renewal such as ERK1/2,FAK, Met, PKC-alpha, SHP2, Smad, STAT1, and STAT3 (Table 3). It is notedherein that DEspR's phosphoproteome overlaps with VEGFR2/VEGF for somesignaling molecules like FAK, ERK1/2, Raf, PKCα[19]. However, thecollective signaling complexes of DEspR/ET1 and DEspR/VEGFsp (Table 3)are quite distinct from that described for VEGFR2/VEGFa [19], thusconfirming non-redundant angiogenesis roles as deduced from null mutantabnormal angiogenesis phenotypes for DEspR [13] and VEGF [20,21] withidentical embryonic lethality between embryonic E10.5 and E12.5 days,although VEGFR2 or Flk1 null mutants died earlier between E8.5-E9.5 days[22].

Discussion

DEspR as a novel target for anti-tumor vascularization therapy. Thedetection of increased DEspR expression in tumor vascular endothelium,in contrast to normal tissue-matched controls, detection of DEspRexpression in both αSMA-negative capillaries/microvessels andαSMA-positive arterioles and arteries in the tumor stroma, andsuccessful inhibition of angiogenesis through DEspR-inhibition alldemonstrate that DEspR is a novel target for therapies aimed at bothtumor angiogenesis and at existing or ‘mature’ tumor microvasculature.More specifically, targeting DEspR on αSMA-positive microvessels canaddress anti-VEGF therapy-resistant tumors which are thought, withoutwishing to be limited or bound by a theory, to have a stromalvasculature no longer dependent on VEGF due their ‘maturation’ as markedby αSMA-positive pericyte sheath or non-dependent on VEGF due to“cooption of existing” microvasculature [2]. Furthermore, combinedtargeting DEspR along with anti-VEGF therapies can address the expectedconcomitant 10-fold increase in VEGFsp that accompanies the observed10-fold increase in VEGF upon anti-VEGF therapy [10], since VEGF andVEGFsp originate from a common propeptide.

Insights from the ligand-specific DEspR phosphoproteome. Given thathypoxia inducible factor-1 alpha (HIF1α) stabilization induces VEGF, andhence VEGFsp, in hypoxia, phosphorylation of BRCA1 and induction of PCNAexpression by VEGFsp-DEspR activation (Table 3), indicates that DEspRcan contribute to the needed DNA repair response activated in hypoxia[24], thus allowing DEspR-positive endothelial and cancer cells toproliferate despite the hypoxic microenvironment, rather than undergohypoxia-induced cell cycle arrest and apoptosis [24,25]. The hepatocytegrowth factor receptor, MET, is induced upon ET1/DEspR stimulation andSmad1/5/9 is phosphorylated upon DEspR/VEGFsp activation, thusindicating a mechanism for crosstalk and/or redundancy amongVEGFsp/DEspR, MET/HGF, and TGFβ/Smad pathways pertinent to angiogenesisin endothelial cells and invasiveness in cancer cells. Importantly,DEspR phosphorylates BRCA1 and STAT3 both of which have been shown tostabilize HIF1α, and along with Raf1, lead to the induction of VEGF, andhence VEGFsp. Furthermore, the phosphorylation of BRCA1 [26] byVEGFsp/DEspR and STAT3 by both ET1/DEspR and VEGFsp/DEspR, can both leadto DEspR-mediated stabilization of HIF1-alpha without the need forhypoxia, leading to constitutive HIF1-α mediated pro-angiogenic andpro-DNA repair response which can contribute to tumor resistance toconventional therapy.

DEspR inhibition as target for dual anti-angiogenesis/anti-cancer cellinvasiveness treatment paradigm. In addition to expression on tumorvascular endothelium, DEspR is expressed in solid tumor epithelial cellsseen in both established cancer cell lines and histology sections ofbreast, pancreatic, lung, stomach, bladder and colon cancers (FIGS.24A-24F, 25A-25F, 26A-26F, and 27A-27F). Just as anti-DEspR inhibitionreduces in vitro angiogenesis (FIGS. 28A-28B), 7C5B2 monoclonalantibody-inhibition decreases tumor cell invasiveness in two aggressivecancer cell lines, breast cancer cell line MDA-MB-231 (and -468) andpancreatic cancer cell line PANC-1 (FIGS. 29A-29C). Thus, targetingDEspR as a receptor involved in both angiogenesis and tumor cellinvasiveness via anti-hDEspR monoclonal antibody -inhibition using thecompositions and methods described herein provides a robust newanti-tumor therapy, and demonstrates the use of the anti-hDEspR 7C5B2monoclonal antibody described herein aas an anti-hDEspR monoclonalantibody-therapeutic precursor.

Furthermore, dual-targeting of angiogenesis and metastasis mechanismscomprise novel methods for next-generation anti-cancer treatmentstrategies [2]. The data described herein demonstrate that targetingDEspR is can be used to achieve a dual-treatment paradigm. The increasedexpression in both pancreatic tumor neovessel and tumor cells, alongwith the inhibition of angiogenesis and pancreatic cancer cell linePANC-1 cell-invasiveness by anti-DEspR inhibition altogether indicatethat anti-DEspR therapy can provide a new treatment approach forpancreatic cancer. The combinatorial anti-angiogenesis andanti-invasiveness caused by DEspR-inhibition, as shown herein, as wellas targeting DEspR for dual tumor endothelial and tumor celltargeted-delivery, can be used, in some embodiments, as a therapeuticbasis for next generation dual anti-tumor/anti-angiogenesis cancertherapies and methods thereof [2].

TABLE 3 Tumor array analysis of DEspR expression in different cancersand cancer cell lines. ↑tumor Cancer vascular Representative Cancer celllines tissue-type endothelium vs cancer ▪DEspR-positive (n) normal types□DEspR-negative Bladder (23) 17/23 Adenocarcinoma ▪*253J BV (74%)Squamous cell ca Transitional cell ca Breast (36) 34/36 Invasive ductalca ▪*MDA-MB-231 (94%) Adenoca ▪*MDA-MB-468 Medullary ca Invasive lobularca Colon (6) 5/6 Adenoca ▪*SW480 Liver (35) 24/35 Hepatocellular ca▪HEP3B (68%) Clear cell ca □HEPG2 Bile duct ca Lung (2) 2/2Adenocarcinoma ▪*NCI-H627 □NCI-H292 Pancreas (6) 6/6 Ductal carcinoma▪*PANC-1 Stomach (2) Primary and in Adenocarcinoma na metastasis to lung*nuclear membrane immunostaining; ca, carcinoma; cancer cell linenomenclature based on ATCC; na, not available on cell-line array, n,number of biopsy cores on tissue array.

TABLE 4 Phosphoproteome of hDEspR upon ET1 and VEGFsp stimulationrespectively. ET1 VEGFsp Pro- Pro- Pro- Protein Name Symbol P*-Site (%CFC) (% CFC) Angiogenesis Cancer Stem cell Breast cancer type 1 BRCA1S1497 32 82 [26] [27] susceptibility protein Cyclin-dependent protein-CDK1/ T14/Y15 53 −16 [28] serine kinase ½ 2 Y1 5 281 −57 Extracellularregulated ERK1/ T202 + 135 −25 [29, 30] [31-33] NSC: [34] protein-serinekinase ½ 2 Y204; CSC: [35] (p44/p42 MAP kinases) T185 + Y187 Focaladhesion protein- FAK S722 55 −38 [36, 37] Metastasis: NSC: [34]tyrosine kinase S732 62 −11 [37,38] CSC: [37] Panspecific 205 0Hepatocyte growth factor Met Panspecific 384 0 [39-41] Metastasis: [43]receptor-tyrosine kinase [42]; Resistance: [39] Proliferating cellnuclear PCNA Panspecific −47 119 [44] antigen Protein-serine kinase C-PKCa T638/T641 137 −17 [45, 46] alpha Protein-serine kinase C- PKCePanspecific 103 −29 [47-50] [50] NSC: epsilon [34]; CSC: [50] Raf1proto-oncogene- Raf1 S259 12 63 [51] [52] encoded protein-serine kinaseSH2 domain-containing Shc1 Y349, 9 97 [53-55] [53, 56, 57] transformingprotein 1 or Y350 ShcA Protein-tyrosine SHP2 S576 14 97 [58-60] [58, 61,62] [61, 63-65] phosphatase 1D SMA- and mothers against Smad S463 + 18147 [30] [66] HSC: [67] decapentaplegic homologs 1/5/9 S465/ 1/5/9S465 + S467 Src proto-oncogene- Src Y529 −20 73 [47] [68-70] encodedprotein-tyrosine Y418 −11 174 kinase Signal transducer and STAT1 S727 86123 Metastasis, CSC: [72] activator of transcription 1 Y701 95 557invasiveness: [71] Signal transducer and STAT3 S727 133 126 [73-75] [74]NSC: [76] activator of transcription 3 Invasiveness: [75] CSC, cancerstem cell; ET1, endothelin 1; hDEspR, human dual endothelin- 1/vascularendothelial growth factor-signal peptide receptor; NSC, neural stemcell; VEGFsp, vascular endothelial growth factor-signal peptide; % CFC,percentage change in treated vs non-treated control averages: % CFC =[Treated − Control]/Control ave] × 100. Phospho-site, phosphorylationsite detected with phosphorylated site-specific antibodies. Datarepresent >50% CFC taken from mean of treated vs control non-treatedduplicates (A, B) with % error range < 20%. % error range = [Treated_(A)− ave]/ave × 100. Kinexus antibody array: phosphoprotein-specific ab todetect phosphorylation changes, and panspecific antibodies to detectexpression changes.

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Example 4 7C5B2 Antibody Sequencing and hDEspR Composite Human AntibodyVariant Generation

Described herein are sequencing results obtained from the monoclonalantibody expressed by the murine hybridoma 7C5B2 (anti-hDEspR), in whichthe heavy and light chain V-region (V_(H) and V_(L)) sequences of the7C5B2 antibody have been determined and exemplary anti-hDEspR compositehuman antibody variants have been designed.

From viable frozen hybridoma cell pellets, RNA was extracted and PCRamplification of antibody specific transcripts was performed afterreverse transcription of mRNA. The nucleotide and amino acid sequencesof the antibody heavy and light chain V-regions were determined and thesequence data was analyzed. Fully humanized antibodies were thendesigned using Composite Human Antibody™ technology, as describedherein.

Methods and Results

RNA Extraction, RT-PCR and Cloning

RNA was extracted from a cell pellet using an RNAqueous®-4PCR kit(Ambion cat. no. AM1914). RT-PCR was performed using degenerate primerpools for murine signal sequences with constant region primers for eachof IgGVH, IgMVH, IgκVL and IgλVE Heavy chain V-region RNA was amplifiedusing a set of six degenerate primer pools (HA to HF) and light chainV-region mRNA was amplified using a set of eight degenerate primerpools, seven for the κ cluster (KA to KG) and one for the λ cluster(LA).

For the heavy chain V-region, amplification products of the expectedsize were obtained from the IgGVH reverse transcription primer andprimer pool HC. For the light chain V-region, amplification productswere obtained from the IgκVL reverse transcription primer and lightchain primer pools KB, KC, KD, and KG (FIG. 33). The PCR products fromeach of the above pools were purified and cloned into a ‘TA’ cloningvector (pGEM (R)-T Easy, Promega cat. no. A1360). Six VH and 24 Vκclones were sequenced.

A single functional VH gene was identified in five clones from pool HCand a single functional Vκ gene sequence was identified in six clonesfrom primer pool KG. The twelve clones from primer pools KB and KC werefound to contain an aberrant transcript (GenBank accession numberM35669) normally associated with the hybridoma fusion partner SP2/0 andthe six clones from pool KD were found to not contain a functional Vκtranscript.

Chimeric Antibody

VH and Vκ (pool KG) genes were PCR amplified using primers thatintroduced flanking restriction enzyme sites for cloning into Antitope'sVH and Vκ chain expression vectors. The VH region was cloned using MluIand HindIII sites, and the VκS region were cloned using BssHII and BamHIrestriction sites. All constructs were confirmed by sequencing.

The chimeric antibody constructs were transiently transfected intoHEK293 cells using calcium phosphate precipitation The transienttransfections were incubated for three days prior to harvestingsupernatants.

Sequence analysis

Analysis of sequences obtained from the hybridoma 7C5B2 is summarized inTable 1. The heavy and light chain V-regions show good homology to theirclosest human germline sequences (64% and 82%, respectively) and theindividual framework sequences have close homologues in the humangermline database.

Design of Composite Human Antibodies

Design of Composite Human Antibody™ Variable Region Sequences

Structural models of the mouse anti-hDEspR 7C5B2antibody V regions wereproduced using Swiss PDB and analysed in order to identify important“constraining” amino acids in the V regions that were likely to beessential for the binding properties of the antibody. Residues containedwithin the CDRs (using Kabat definition) together with a number offramework residues were considered to be important. Both the VH and Vκsequences of anti-hDEspR contain typical framework residues and the CDR1, 2 and 3 motifs are comparable to many murine antibodies.

From the above analysis, it was considered that composite humansequences of anti-hDEspR could be created with a wide latitude ofalternatives outside of CDRs but with only a narrow menu of possiblealternative residues within the CDR sequences. Analysis indicated thatcorresponding sequence segments from several human antibodies could becombined to create CDRs similar or identical to those in the murinesequences. For regions outside of and flanking the CDRs, a wideselection of human sequence segments were identified as components ofthe novel Composite Human Antibody™ V regions described herein (seeTable 1).

Design of Variants

Based upon the above analysis, a large preliminary set of sequencesegments that could be used to create anti-hDEspR Composite HumanAntibody™ variants were selected and analysed using iTope™ technologyfor in silico analysis of peptide binding to human MHC class II alleles(Perry et al 2008), and using the TCED™ (T Cell Epitope Database) ofknown antibody sequence-related T cell epitopes (Bryson et al 2010).Sequence segments that were identified as significant non-human germlinebinders to human MHC class II or that scored significant hits againstthe TCED™ were discarded. This resulted in a reduced set of segments,and combinations of these were again analysed, as above, to ensure thatthe junctions between segments did not contain potential T cellepitopes.

Selected segments were then combined to produce heavy and light chain Vregion sequences for synthesis. For anti-hDEspR, five VH chains (SEQ IDNO: 13-SEQ ID NO: 17) and two Vκ chains (SEQ ID NO: 18 and SEQ ID NO:19) were designed with sequences as detailed herein.

We claim:
 1. An isolated antibody or antigen-binding fragment thereof that specifically binds DEspR (dual endothelin/VEGF signal peptide receptor) of SEQ ID NO: 1 and comprises a V_(H) domain comprising a heavy chain CDR1 having the amino acid sequence of SEQ ID NO: 5; a heavy chain CDR2 having the amino acid sequence of SEQ ID NO: 6; and a heavy chain CDR3 having the amino acid sequence of SEQ ID NO:7 and a V_(L) domain comprising a light chain CDR1 having the amino acid sequence of SEQ ID NO: 10; a light chain CDR2 having the amino acid sequence of SEQ ID NO: 11; and a light chain CDR3 having the amino acid sequence of SEQ ID NO:
 12. 2. The isolated antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is a DEspR antagonist.
 3. The isolated antibody or antigen-binding fragment thereof of claim 1, that specifically binds to an epitope of DEspR of comprising amino acids 1-9 of SEQ ID NO:
 1. 4. An isolated antibody or antigen-binding fragment thereof that specifically binds DEspR (dual endothelin/VEGF signal peptide receptor) of SEQ ID NO: 1 comprising a V_(H) domain comprising the amino acid sequence selected from SEQ ID NO: 4 and any one of SEQ ID NOs: 13-17.
 5. An isolated antibody or antigen-binding fragment thereof that specifically binds DEspR (dual endothelin/VEGF signal peptide receptor) of SEQ ID NO: 1 comprising a V_(H) domain comprising a heavy chain CDR1 of SEQ ID NO: 5; a heavy chain CDR2 of SEQ ID NO: 6; and a heavy chain CDR3 of SEQ ID NO: 7 and a V_(L) domain comprising an amino acid sequence selected from SEQ ID NO:9, SEQ ID NO: 18, and SEQ ID NO:
 19. 6. The antibody or antigen-binding fragment thereof according to claim 1, comprising two copies of said V_(H) domain.
 7. The antibody or antigen-binding fragment thereof according to claim 1, further comprising an agent conjugated to the antibody or fragment thereof to form an immunoconjugate specific for DEspR.
 8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the agent conjugated to the antibody or antibody fragment thereof is a chemotherapeutic agent, a toxin, a radioactive isotope, a small molecule, an siRNA, a nanoparticle, or a microbubble.
 9. The antibody or antigen-binding fragment thereof according to claim 4, comprising two copies of said V_(H) domain.
 10. A composition comprising the antibody or antigen-binding fragment of claim 1, and a pharmaceutically acceptable excipient, diluent, or carrier.
 11. The antibody or antigen-binding fragment thereof according to claim 4, wherein the antibody is a humanized antibody or antigen-binding fragment thereof, or a composite antibody or antigen-binding fragment thereof.
 12. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody is a humanized antibody or antigen-binding fragment thereof, or a composite antibody or antigen-binding fragment thereof.
 13. The antibody or antigen-binding fragment thereof according to claim 4, comprising two copies of said V_(H) domain.
 14. The antibody or antigen-binding fragment thereof according to claim 4, further comprising an agent conjugated to the antibody or fragment thereof to form an immunoconjugate specific for DEspR.
 15. The antibody or antigen-binding fragment thereof according to claim 14, wherein the agent conjugated to the antibody or antibody fragment thereof is a chemotherapeutic agent, a toxin, a radioactive isotope, a small molecule, an siRNA, a nanoparticle, or a microbubble.
 16. A composition comprising the antibody or antigen-binding fragment of claim 4, and a pharmaceutically acceptable excipient, diluent, or carrier.
 17. The antibody or antigen-binding fragment thereof according to claim 5, further comprising an agent conjugated to the antibody or fragment thereof to form an immunoconjugate specific for DEspR.
 18. The antibody or antigen-binding fragment thereof according to claim 17, wherein the agent conjugated to the antibody or antibody fragment thereof is a chemotherapeutic agent, a toxin, a radioactive isotope, a small molecule, an siRNA, a nanoparticle, or a microbubble.
 19. The antibody or antigen-binding fragment thereof according to claim 5, comprising two copies of said V_(H) domain and said V_(L)domain.
 20. A composition comprising the antibody or antigen-binding fragment of claim 5, and a pharmaceutically acceptable excipient, diluent, or carrier. 